EP2166085A1 - Cellules modifiées bivalentes - Google Patents

Cellules modifiées bivalentes Download PDF

Info

Publication number
EP2166085A1
EP2166085A1 EP08397517A EP08397517A EP2166085A1 EP 2166085 A1 EP2166085 A1 EP 2166085A1 EP 08397517 A EP08397517 A EP 08397517A EP 08397517 A EP08397517 A EP 08397517A EP 2166085 A1 EP2166085 A1 EP 2166085A1
Authority
EP
European Patent Office
Prior art keywords
cells
cell
enzyme
glycan
preferred
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP08397517A
Other languages
German (de)
English (en)
Inventor
Johanna Nystedt
Heidi Anderson
Leena Valmu
Jari Natunen
Tero Satomaa
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Suomen Punainen Risti Veripalvelu
Glykos Finland Ltd
Original Assignee
Suomen Punainen Risti Veripalvelu
Glykos Finland Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Suomen Punainen Risti Veripalvelu, Glykos Finland Ltd filed Critical Suomen Punainen Risti Veripalvelu
Priority to EP08397517A priority Critical patent/EP2166085A1/fr
Priority to US13/054,541 priority patent/US9234169B2/en
Priority to AU2009272654A priority patent/AU2009272654B2/en
Priority to EP09797548A priority patent/EP2318540A4/fr
Priority to PCT/FI2009/050628 priority patent/WO2010007214A1/fr
Publication of EP2166085A1 publication Critical patent/EP2166085A1/fr
Priority to US14/982,800 priority patent/US20160215256A1/en
Withdrawn legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0006—Modification of the membrane of cells, e.g. cell decoration
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/005—Glycopeptides, glycoproteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases [EC 2.]
    • C12N2501/724—Glycosyltransferases (EC 2.4.)

Definitions

  • the invention is directed to a method and composition to control and modify the status of human stem cells by changing their glycosylation, in particular sialylation and fucosylation, levels in a reaction condition where divalent cations are present and cells are kept non-adherent.
  • the invention is further directed to novel stem cells, the glycosylation of which has been specifically altered.
  • PCT/FI2006/050323 published 18.1.2007 , describes a modification of glycosylation of stem cells.
  • the examples show reaction of human cord blood mononuclear cells, comprising hematopoietic stem cells, with sialyltransferase and fucosyltransferases in MOPS buffer and 150 mM NaCl and by sialidases in acetate buffer.
  • WO 2008/011094 also by Sackstein describes the use of cytokines to stimulate glycosylation enzymes on hematopoietic stem cells.
  • the present invention is directed to the use of non-toxic divalent cations, small molecules and supporting non-glycoprotein albumin in context of in vitro modification of especially adherent cell. It is realized that activation of cells by cytokines may also produce undesired differentiation or cell activation leading to negative activities in context of in vivo or ex vivo uses of the cell.
  • PCT/FI2008/050015 filed 18.1.2008 , by the present inventors indicates that the reactions even with the adherent cells can be performed in the presence of Mg 2+ , which would be preferred for the activity of modification enzymes, especially sialyltransferase and fucosyltransferase.
  • the example includes reactions to mesenchymal stem cells in ⁇ -MEM, which contains also other factors revealed useful by present invention.
  • the present invention revealed that use of both Mg 2+ and Ca 2+ ions preferably with supporting factors is especially useful for producing optimally viable cells.
  • the preferred conditions further include conditions to prevent aggregation and surface binding of cells, especially preferred shear force conditions.
  • PCT/FI2008/050015 filed 18.1.2008 , includes enzyme tagging technologies.
  • the present invention provides specific forms and methods for the tagging, including glycan tagging with multiple tags and limited lysine-specific tagging by biotinylation.
  • the present invention is further directed to the use of strong protease conditions producing unicellular cell suspension. It is realized that this is needed to produce effective glycosylation and optimal unicellular product for subsequent in vivo use. It is realized that the cell population produced under the Sackstein conditions with a low amount of protease might not result in a unicellular preparation.
  • the present invention further provides methods for specific tagging and washing cells with glycosyltransferase inhibitors to remove the enzymes from cell preparations after modification reactions.
  • the invention provides a novel method or composition for modification of glycosylation of human stem cells.
  • the invention revealed that it is possible to modify glycosylation of cells, when the modification is performed in presence of divalent cations Mg 2+ and/or Ca 2+ .
  • the invention is especially directed to glycosyltransferase modifications by sialyl- and fucosyltransferases.
  • the invention revealed specific reagents for removal of the modification enzymes from reaction mixtures including site specific tags of enzymes and specific substrate inhibitors.
  • the invention is directed to a novel cell population and to a method for its production.
  • the cell population is derived from human stem cells and the cell population comprises in vitro enzymatically modified glycosylation produced by a method comprising enzymatic in vitro glycan modification in the presence of divalent cations Mg 2+ and Ca 2+ and adherence of the cells is inhibited by shear force.
  • the modification enzyme is removed from the cell preparation by using at least one reagent selected from the group a glycan linked tag, not essentially reducing the enzymatic activity of the protein; a protein specifically linked tag, not essentially reducing the enzymatic activity of the enzyme protein; a specific substrate inhibitor binding effective to the active site of the enzyme and releasing the enzyme from the cells, preferably being acceptor substrate analog of glycosyltransferase or substrate analog for protease.
  • a glycan linked tag not essentially reducing the enzymatic activity of the protein
  • a protein specifically linked tag not essentially reducing the enzymatic activity of the enzyme protein
  • a specific substrate inhibitor binding effective to the active site of the enzyme and releasing the enzyme from the cells preferably being acceptor substrate analog of glycosyltransferase or substrate analog for protease.
  • the novel cells are preferably prepared or derived from cord blood or bone marrow derived mesenchymal stem cells or other mesenchymal stem cells.
  • the cells are prepared or derived from isolated cord blood cells and in another embodiment from bone marrow derived cells.
  • the invention is specifically directed to adherent cells. It is realized that several modifications can change also other cell types adherent.
  • the invention is directed to modification of stem cells by the methods according to the present invention, when the adhesion of the cells is increased.
  • the novel cell population is preferably produced by a method, wherein the cells are modified in the presence of Mg 2+ . It was realized that Mg 2+ would support the glycosylation reactions.
  • the novel cell population is preferably produced by method, wherein the cells are modified in the presence of Ca 2+ .
  • the invention revealed that presence of Ca 2+ is useful for maintaining the cell morphology. Even more preferably the cells are modified in the presence of both Ca 2+ and Mg 2+ .
  • the invention is further directed to cell population and its production, wherein the cells are modified in the presence of additional supporting factors selected from the group consisting of vitamins, energy and structural nutrients, and physiological salt.
  • novel cells are modified in the presence of non-glycoprotein.
  • the cells are modified in the presence of at least one cell supporting materials, preferably all additional supporting factors selected from the group consisting of
  • the invention is specially directed to the combination of the factors, which is especially useful for maintaining the cells during the modification reactions.
  • both Mg 2+ and Ca 2+ ions are used in a concentration of at least 0.05 mM, even more preferably 0.5 mM and most preferably at least 1 mM and less than 10 mM.
  • the preferred concentrations of the non-toxic divalent cations are close to physiological concentration, preferably between about 0.5 mM and 1.0 mM, more preferably about 0.8 mM for Ca 2+ , and preferred Mg 2+ concentration is between about 1.5 and 2 mM, more preferably about 1.8 mM.
  • the ions and optionally additional ions are used in concentrations similar to those in the ⁇ -MEM medium, also referred to as the minimal essential medium (Nature 1971, Catalog of Gibco/Invitrogen®).
  • the invention is especially directed to novel cell populations produced according to the invention, wherein the cells are adherent cells detached from cell culture surface by protease treatment.
  • the cells are preferably detached under condition producing unicellular cell suspension.
  • the preferred trypsin condition includes trypsin concentrations from about 0.01 mg/ml to about 0.5 mg/ml, wherein 1 mg corresponds to about 10 000 BAEE units of trypsin, as defined by the producer of the trypsin (Invitrogen).
  • the trypsin is preferably from bovine (product number 25200, Invitrogen).
  • the reaction is preferably short, i.e. from about 0.5 min to about 5 min, more preferably about 3 min.
  • the cell population is produced from strongly adherent cells and protease is trypsin which is used in a condition equivalent of trypsin concentration of 0.075 mg/ml, more preferably 1 mg/ml, most preferably 0.25 mg/ml for about three minutes.
  • the invention is especially directed to mesenchymal cells, which are released from adherent cell culture by a protease, preferably trypsin under optimal conditions (Example 3).
  • the invention is directed to the release of adherent mesenchymal stem cells from cell culture support under proteolysis condition producing unicellular cell preparation.
  • the invention further revealed that the unicellular cell suspension is effectively modified in glycomodification reactions (Examples 2 and 3).
  • the invention revealed that it is useful to end the protease reaction by inhibitor molecules.
  • the protease reaction is ended by adding an inhibitor of protease, preferably substrate inhibitor which releases the protease from the cell surface (Example 3).
  • the invention is further directed to the novel cells and their production method, wherein the protease is effectively removed from cell surface of the modified cells, preferably using a covalent tag attached to the protease and/or a substrate inhibitor of the protease, which is optionally tagged.
  • the protease enzyme and/or its substrate inhibitor are preferably tagged by at least one covalent tagging method selected from the group consisting of protein amine tagging, glycan tagging, N-terminal tagging of protein or peptide and C-terminal tagging of protein or peptide.
  • the invention is further directed to a method for removal of the protease and/or protease inhibitor, wherein the protease and/or substrate inhibitor of the protease is tagged and the inhibitor and protease are effectively removed from the surface of the modified cells by using an adsorbent binding the tag (Example 5).
  • the protease is tagged and high-affinity inhibitor peptide or protein is used.
  • the invention revealed that it is possible to modify adherent cells, preferably adherent stem cells and most preferably mesenchymal stem cells in presence of divalent cations, when the divalent cation is non-toxic.
  • the non-toxic divalent cation is preferably not toxic as high micromolar or millimomolar concentration.
  • An example of a toxic divalent cation is Mn 2+ cation.
  • the present method is in the contrast to the method of Sackstein wherein all divalent cations were omitted.
  • the preferred non-glycoproteins of present invention do not include cytokines according to WO 2008/011094 .
  • the present invention reveals that in the presence of specific divalent cations mesenchymal cells are more viable and can be effectively glycosylated by glycosyltransferases, especially by sialyltransferases and fucosyltransferases.
  • the reaction media lacking divalent cations is not optimal for mesenchymal stem cells.
  • the cells appear viable their ability to recover from the stress due to proteolytic treatment is reduced and the cells tend to adhere to aggregates which reduce both their viability and their glycans accessibility to be modified.
  • the invention revealed that aggregation and surface attachment occurs in the presence of non-toxic divalent cations.
  • it is possible to recover the adherent mesenchymal stem cells.
  • the presence of the divalent cations provided 100 % viability. This is a clear benefit as the dead cells may cause undesired immunological and other reactions in therapeutic use.
  • resuspension of the cells prevented aggregation and surface adhesion, hence, leading to a clearly increased yield of the cells (Example 2).
  • the conditions in the Sackstein-process without divalent cations can be regarded as non optimal to the activity for many fucosyltransferases and sialyltransferases.
  • the presence of divalent cations is especially preferred for fucosyltransferase reactions.
  • Presence of divalent cations is especially preferred for fucosyltransferases FUC-TIII-VII, more preferably for Fuc-TVI and Fuc-TVII.
  • Sialyltransferases are also known to function optimally with divalent cations.
  • both Mg 2+ and Ca 2+ ions are used with at least 0.05 mM, more preferably at least 0.1 mM, even more preferably 0.5 mM and most preferably at least 1 mM and less than 10 mM. More preferably the ions are use as physiological concentrations, preferably similar as in ⁇ -MEM.
  • the invention revealed that the novel cells produced by the present invention are maintained in mononuclear cell-like morphology while the conditions without non-toxic divalent cations caused granularity of the cells.
  • the invention is especially directed to the novel cell population, wherein the cells in suspension are essentially mononuclear cell-like cells without granularity (Example 2).
  • the invention is directed to glycomodification of adherent cells more preferably mesenchymal stem cells in the presence of non-glycoproteins including non-glycosylated or non-glycosylable protein.
  • the non-glycoprotein is protein, which does not contain substantial amount of acceptor glycans for modification enzymes, such as terminal Gal, lactosamine(s), GalNAc, or subterminal GlcNAc.
  • the preferred non-glycoproteins include non-glycosylated proteins such as serum albumin, which does not contain glycosylations sites, in a preferred embodiment bovine serum albumin and non-glycosylated isoforms of human serum albumin.
  • non-glycosylated human serum albumin is purified from a blood-derived human serum albumin by affinity chromatography by a reagent binding to the glycans of glycosylated isoform of human serum albumin.
  • residual glycosylation is removed by a chemical or enzymatic method.
  • the invention revealed that it is possible to perform enzymatic glycomodification in the presence of "supporting factors", meaning here, for example, (i) vitamins, including enzyme cofactors, (ii) nutrients with energy functions, or (iii) nutrients with structural functions, all preferably in concentrations similar to those in the ⁇ -MEM medium.
  • supporting factors meaning here, for example, (i) vitamins, including enzyme cofactors, (ii) nutrients with energy functions, or (iii) nutrients with structural functions, all preferably in concentrations similar to those in the ⁇ -MEM medium.
  • the reaction is performed in the presence of metabolic factors as present in a serum-free cell culture medium.
  • the cell culture medium is ⁇ -MEM for human mesenchymal stem cells.
  • the medium contains only non-glycoproteins according to the invention and no other acceptor glycans or oligosaccharides, which would affect the activity of the modification enzymes.
  • the present cells are adherent cells and modified in a condition preventing adhesion of the cells, especially their clustering and surface adherence.
  • conditions are shear force involving conditions. It is further realized that the prevention of clustering may be obtained by various chemical factors.
  • the shear force to the cells is provided by tubing or tubing with a changing flow velocity that detaches the cells, or by tubing comprising a narrowing that provides turning flow direction and a swirl or swirls that detach cells from each other, or by applying a flow of liquid with swirls on cells.
  • the administration of the shear force preferably includes providing a certain flow accelerating and decelerating flow velocity to the cells in the tubing (Example 2).
  • the preferred method for the production of the cells included a modification step, wherein the cells are modified for at least 0.5 hours, more preferably over 1 hour, even more preferably over 1.5 hours and even more preferably 2 hours or longer, such as 2 to 4 hours. It is realized that present reaction conditions allow maintaining the reactions over one hour, while an incubation of stem cells in salt solutions without divalent cations leads to apparently harmful morphological changes already within one hour (Example 1).
  • Glycan modification including shear force
  • the invention is in a preferred embodiment directed to modification of adherent cells including at least one step of exposing cells to shear force. It is realized that the cells may adhere to each other and/or cell culture support during the modification reagent, and this would reduce the effect of the cell modification reaction(s) and may diminish their viability (Example 2).
  • More preferably cells are exposed to shear forces for maintaining unicellular cell state in presence of divalent cations, preferably Ca 2+ and/or Mg 2+ . It is realized that the presence of divalent cations increase cell adhesion by the receptors dependent on divalent cations.
  • divalent cations preferably Ca 2+ and/or Mg 2+ .
  • the cells are exposed to the shear stress for at least one period during the modification reaction for time allowing suspension of cell starting to adhere to suspend to unicellular composition.
  • the shear stress is administered for at least three, and more preferably at least four periods.
  • the length of the period is at least 5 seconds, more preferably at least 15 seconds, and most preferably at least 30 seconds.
  • the shear force is administered by mechanical agitation of the cell preparation in solution.
  • the mechanical agitation includes administration of the cells through a hole or tube, which is narrow enough to provide shear stress to cells. More preferably the shear stress is administered by sucking cells into a tip structure similar to pipette tip, preferably a tip of a Biohit m1000 pipette with Art 1000E filter tip (Example 2).
  • the invention revealed that optimal reaction can be obtained by using proteolytic enzyme in relatively large concentrations and amount per cells (Example 3).
  • the present conditions may be obvious for maintaining metabolic activity of the cells, but the increase of sialyl-Lewis x structures (Example 1), especially the core 2 sialyl-Lewis x epitope was not expected. It is realized that the spontaneous cell surface modification appears partially be related to glycoprotein recovery on cells, furthermore the present media is considered to support the machinery for endogeneous glycosylation.
  • the present invention is further directed to use of the spontaneous modifications together with in vitro modification of the cells.
  • mesenchymal stem cells are modified to increase sialylation and/or fucosylation as a combination method, preferably by preferred transferases for sialyl-LacNAc synthesis and fucosylation such as STGalIII and Fuc-TVI.
  • Preferred fucosyltransferase conditions include about 4 mU (especially for Fuc-TVI, Calbiochem enzyme and units, fresh enzyme) per 3 million cells, or from 0.5 to 5 mU per million cells, more preferably 0.75-3 mU, and most preferably 1-2 mU per million cells.
  • the preferred range depends on the status of the enzyme (dacays during storage) and status and type of the cells.
  • the preferred reaction temperature is about 37°C, preferably between 33-40°C and more preferably 35-39°C.
  • the preferred reaction times varies from 0.5 to 6 hours preferably between 1-6 hours, more preferably between 2-6 hours, even more preferably between 3-5.5 hours and in a preferred embodiment about 4 hours (3.5-4.5 hours). It is realized that increasing the enzyme amount reduces reaction time needed.
  • Preferred sialyltransferase conditions includes about 50 mU (especially for ⁇ 2,3-(N)-Sialyltransferase (Calbiochem), Calbiochem enzyme and units, fresh enzyme) per 1 million cells, or from 5 to 200 mU per million cells, more preferably 10-150 mU, and most preferably 25-75 mU per million cells.
  • the preferred range depends on the status of the enzyme (dacays during storage) and status and type of the cells.
  • the preferred reaction times varies from 0.5 to 6 hours preferably between 1-6 hours, more preferably between 2-6 hours, even more preferably between 3-5.5 hours and in a preferred embodiment about 4 hours (3.5-4.5 hours).
  • the preferred reaction temperature is about 37°C, preferably between 33-40°C and more preferably 35-39°C. It is realized that increasing the enzyme amount reduces reaction time needed.
  • the invention is especially directed to modification of stem cells especially mesenchymal stem cells wherein the cells have unusually low sialylation levels.
  • the cells with low sialylation comprise more than 30 % of N-glycans in non-sialylated form.
  • the mesenchymal stem cell with low sialylation is a bone marrow derived mesenchymal stem cell (Example 6, Example 9).
  • the present invention revealed that it is possible to glycosylate cells, preferably to sialylate cells to over 50% level of available free sialylation sites on N-glycans (when calculated based on the disappearance of the sialylation sites).
  • the invention is directed to sialylation by single sialyltransferase to level over 60%, more preferably over 70%, even more preferably over 75%, even more preferably over 80%, or at least 83%, and most preferably over 85%.
  • the invention is further directed to a novel mesenchymal stem cell population comprising increased sialylation of over 60%, more preferably over 70%, even more preferably over 75%, even more preferably over 80%, or at least 83%, and most preferably over 85%.
  • the cell population is preferably derived from human cord blood or bone marrow.
  • the invention is directed to the novel cells, wherein the cells are at least 98% viable as indicated by intact plasma membrane, most preferably over 99% as indicated by intact plasma membrane.
  • the invention revealed that under the preferred cell modification condition, including e.g. non-toxic cations, leads to highly viable cells, especially, when the cells are produced under conditions in which adherence is actively inhibited by shear force.
  • the preferred viable mesenchymal cells according to the invention have intact plasma membrane (Example 2).
  • control cells did suffer from the incubation in the buffer without the beneficial reagent according to the present invention. It is realized that though the control cells have mostly intact membranes their overall viability and normal cell status is compromised.
  • the invention revealed referred markers of novel glycan modified cell populations, preferably protease produced glycan modified cell populations.
  • the invention is especially directed to in vitro fucosylation methods, when fucosylation increases amount of core 2 sLex, especially epitope similar to one recognized by antibody CHO-131. This structure is effectively formed under present cell modification conditions.
  • the optimal production is obtained by spontaneous regeneration of glycosylation, preferably fucosylation by endogenous fucosyltransferase, and/or by in vitro glycosylation preferably fucosylation or fucosylation and sialylation, preferably by both.
  • the invention revealed that the sialylation and fucosylation reactions according to the invention increased specific high MAA subpopulation of the mesenchymal cells.
  • MAA recognizes a specific highly ⁇ 3-sialylated in the novel cell populations (Example 1).
  • the fucosylation and sialylation reactions further provide effectively the production of sialyl-CD15 and core 2 sLex (antibody CHO-131) epitopes.
  • the invention is in a preferred embodiment directed to modification of specific mesenchymal stem cell preparations, in a preferred embodiment produced by protease, such as trypsin treatment wherein the amount of certain glycan structures are reduced (Example 1): the amount of N-glycans, which can be modified by sialylation on cell surface, or the amount of specific non-N-glycan sLex (sialyl-Lewis x) epitope, preferably O-glycan associated glycans, especially GF526 epitope (antibody clone CHO-131).
  • the GF526 epitope is core 2 O-glycan, comprising sLex on ⁇ 6-linked arm.
  • the structure is in other context associated with PSGL-1 protein.
  • the invention revealed that it is possible to change quantitatively the sialylation levels of human cells.
  • the signals of monosialylated and disialylated sialic acids of biantennary N-glycan cores were measured by MALDI-TOF mass spectrometry of released non-modified N-glycans. It was observed that the sialylation levels of the N-glycans of cells could be increased at least by 15% units and even by about 20% or 25% by sialylation of the cells by sialyltransferase enzyme. It was also observed that the sialylation levels of the N-glycans of cells could be decreased at least by 15% units and even by about 20% or 25% by sialylation of the cells by sialylidase (neuraminidase) enzyme.
  • the invention is especially directed to cell populations of quantitatively increased and decreased sialylation levels.
  • the invention revealed furthermore that the ⁇ 3-sialylated cells can be fucosylated to produce cells increased in their sialylated and fucosylated levels, comprising sialyl-Lewis x Neu5Ac ⁇ 3Gal ⁇ 4(Fuc ⁇ 3)GlcNAc (sLex) and related structures. It is realized that sLex content can be further increased by first resialylating the cells and thus reducing ⁇ 6-sialylated structures blocking sites. Such sialyl-Lewis x cells are especially useful for in vivo targeting as the structures produced in low amounts from endogenous Neu5Ac ⁇ 3Gal ⁇ 4GlcNAc can redirect the cells (Xia et al. 2004) (Example 1).
  • novel cells produced by the invention are useful for in vivo targeting in human and animal trials, or potentially in therapeutic applications.
  • the invention is in a specific embodiment directed to altering surface adhesion of glycan modified cells. It is thus realized that glycan modification can be used to change adherence properties of cells.
  • the change of adherence properties is used for e.g.
  • the invention is in a specific embodiment directed to cell culture of glycan modified cells with altered adherence properties.
  • the cells are cultured after modification, meaning increase of sialylation by sialyltransferase or reduction of sialic acids by sialidase enzyme.
  • the invention is directed to modification of naturally adherent cells, preferably mesenchymal stem cells.
  • the invention revealed novel effective methods for modifying cells by glycosyl modifying enzymes such as glycosidases and/or glycosyltransferases, when the enzymes are removed from the cell preparations.
  • glycosyl modifying enzymes such as glycosidases and/or glycosyltransferases
  • the invention is especially directed to use of specific tag-structures for the removal of the enzymes from the cells (Example 5).
  • enzymes bind cells by their carbohydrate binding sites such as catalytic sites.
  • the enzymes are removed by incubating the cells with an inhibitor of the enzymes, preferably an inhibitor binding to the catalytic carbohydrate recognizing site of the enzyme.
  • Preferred inhibitors include monosaccharides and monosaccharide glycosides such as methyl and ethyl glycosides and more specific inhibitors, which may be designed based on the catalytic site as transition state inhibitors.
  • Preferred inhibitors for sialidases include competitive low activity inhibitors such as sialic acid, and modified or low cost competing substrates such as NeuAc ⁇ OMe, NeuNAc ⁇ OEt, sialyl-Lactoses available e.g.
  • bovine milk or polysialic acid available from bacteria E. coli , colomnic acid
  • higher activity inhibitors such as NeuAc2en (NeuNAc with double bond between 2- and 3-positions) or e.g. higher activity inhibitors specific for limited number of enzymes such as influenza virus neuraminidase inhibitors: Tamiflu (oseltamivir, Roche) or Zanamivir (GSK).
  • the amount of enzyme inhibitor needed can be estimated by inhibition constants.
  • Competitive monosaccharide glycoside or oligosaccharide inhibitors with low millimolar inhibition (or binding constants) are typically needed in amounts of several fold or order of magnitude larger amounts than the inhibition constant.
  • Typical concentrations for the low affinity inhibitors are of about 1- 500 mM, more preferably 1 - 250 mM, and more preferably 2 - 100 mM, or 2 to 50 mM, even more preferably from about 2 mM to 20 mM. The lower ranges are preferred to maintain the stability and osmotic condition of the cells stable.
  • Typical concentrations for higher affinity inhibitors are from about 1 pM to about 10 mM, depending about the affinity constants.
  • Preferred concentrations for low range micromolar inhibitor are between 10 - 1000 micromolar. Suitable inhibition concentrations are available from literature.
  • the invention is directed for removing modification enzyme from modified cells involving a step of incubation of the cells with an inhibitor or substrate of the enzyme.
  • the method preferably further comprises steps of washing cells with a suitable solution such as PBS (phosphate buffered saline) or other solution suitable, optionally containing additional amount of inhibitor, and preferably a step of final washing with the solution not comprising the inhibitor.
  • a suitable solution such as PBS (phosphate buffered saline) or other solution suitable, optionally containing additional amount of inhibitor, and preferably a step of final washing with the solution not comprising the inhibitor.
  • the invention is directed to removal of the enzyme by a combination of the enzyme tagging with the use of the inhibitors.
  • the invention is specifically directed to desialylation methods for modification of human cord blood cells.
  • the cord blood cells are clearly different of other cell types and no desialylation methods have previously been developed for these cells. Due to cell specific differences any quantitative desialylation methods cannot be generalized from one cell population to another. Thus, any results and data demonstrated by other investigators using other cell types are not applicable to cord blood.
  • the present invention is further directed to desialylation modifications of any human stem cell or cord blood cell subpopulation.
  • the invention is preferably directed to linkage specific ⁇ 3-desialylation of the preferred structures according to the invention without interfering with the other sialylated structures according to the present invention.
  • the invention is further directed to simultaneous desialylation ⁇ 3- and ⁇ 6-sialylated structures according to the present invention.
  • the present invention is directed to desialylation when both NeuAc and NeuGc are quantitatively removed from cell surface, preferably from the preferred structures according to the present invention.
  • the present invention is specifically directed to the removal of NeuGc from preferred cell populations, most preferably cord blood and stem cell populations and from the preferred structures according to the present invention.
  • the inventors revealed that it is possible to produce controlled cell surface glycosylation modifications on the preferred cells according to the invention.
  • the present invention is directed to cell modifications by sialyltransferases and fucosyltransferases.
  • Two most preferred transfer reactions according to the invention are ⁇ 3-modification reactions such as ⁇ 3-sialylation and ⁇ 3-fucosylations. When combined these reactions can be used to produce important cell adhesion structures which are sialylated and fucosylated N-acetyllactosamines such as sialyl-Lewis x (sLex).
  • the inventors of the present invention further revealed effective modification of the preferred cells according to the present inventions by sialylation, in a preferred embodiment by ⁇ 3-sialylation.
  • the prior art data cited above does not indicate the specific modifications according to the present invention to cells from early human blood, preferably cord blood, to cultured mesenchymal stem cells, or to cultured embryonal type cells.
  • the present invention is specifically directed to sialyltransferase reactions towards these cell types.
  • the invention is directed to sialyltransferase catalyzed transfer of a natural sialic acid, preferably NeuAc, NeuGc or Neu-O-Ac, from CMP-sialic acid to target cells.
  • SA is a sialic acid, preferably a natural sialic acid, preferably NeuAc, NeuGc or Neu-O-Ac and the reaction is catalysed by a sialyltransferase enzyme preferably by an ⁇ 3-sialyltransferase and the target cell is a cultured stem cell or early human blood cell (cord blood cell).
  • the sialic acid is transferred to at least one N-glycan structure on the cell surface, preferably to form a preferred sialylated structure according to the invention.
  • the invention is specifically directed to selection of a cell population from the preferred cell population according to the present invention, when the cell population demonstrate increased amount of ⁇ 3-sialylation when compared with the baseline cell populations.
  • human cord blood in general is highly ⁇ 6-sialylated and thus not a good target for ⁇ 3/4-fucosylation reactions, especially for reactions directed to production of selectin ligand structures.
  • the present invention is preferably directed to fucosylation after ⁇ 3-sialylation of cells, preferably the preferred cells according to the invention.
  • the invention describes for the first time combined reaction by two glycosyltransferases for the production of specific terminal epitopes comprising two different monosaccharide types on cell surfaces.
  • Present invention is specifically directed to methods for sialylation to produce preferred structures according to the present invention from the surfaces of preferred cells.
  • the present invention is specifically directed to production preferred NeuGc- and NeuAc-structures.
  • the invention is directed to production of potentially in vivo harmful structures on cells surfaces, e.g. for control materials with regard to cell labelling.
  • the invention is further directed to production of specific preferred terminal structure types, preferably ⁇ 3-and ⁇ 6-sialylated structures, and specifically NeuAc- and NeuGc-structures for studies of biological activities of the cells.
  • the present invention is further directed to preferred methods for the quantitative verification of the sialylation by the preferred analysis methods according to the present invention.
  • the present invention is further directed to linkage specific sialylation and analysis of the linkage specific sialylation on the preferred carbohydrate structures using analytical methods according to the present invention.
  • the invention is preferably directed to linkage specific ⁇ 3-sialylation of the preferred structures according to the invention without interfering with the other sialylated structures according to the present invention.
  • the invention is preferably directed to linkage specific ⁇ 6-sialylation of the preferred structures according to the invention without interfering with the other sialylated structures according to the present invention.
  • the invention is further directed to simultaneous sialylation ⁇ 3- and ⁇ 6-sialylated structures according to the present invention.
  • the present invention is further directed for the production of preferred relation of ⁇ 3- and ⁇ 6-sialylated structures, preferably in single reaction with two sialyl-transferases.
  • the present invention is directed to sialylation when either NeuAc or NeuGc are quantitatively synthesized to the cell surface, preferably on the preferred structures according to the present invention. Furthermore the invention is directed to sialylation when both NeuAc and NeuGc are, preferably quantitatively, transferred to acceptor sites on the cell surface.
  • the present invention is specifically directed to the removal of NeuGc from preferred cell populations, most preferably cord blood cell populations and from the preferred structures according to the present invention, and resialylation with NeuAc.
  • the invention is further directed to preferred methods according to the present invention for verification of removal of NeuGc, and resialylation with NeuAc, preferably quantitative verification and more preferably verification performed by mass spectrometry with regard to the preferred structures.
  • the present invention is further directed to cell modification according to the invention, preferably desialylation or sialylation of the cells according to the invention, when the sialidase reagent is a controlled reagent with regard of presence of carbohydrate material.
  • the preferred processes according to the invention comprise of the step of removal of the enzymes from the cell preparations, preferably the sialyl modification enzymes according to the invention. Most preferably the enzymes are removed from a cell population aimed for therapeutic use.
  • the enzyme proteins are usually antigenic, especially when these are from non-mammalian origin. If the material is not of human origin its glycosylation likely increases the antigenicity of the material.
  • glycosylation has major differences with human glycosylation
  • preferred examples of largely different glycosylations include: prokaryotic glycosylation, plant type glycosylation, yeast or fungal glycosylation, mammalian/animal glycosylation with Gal ⁇ 3Gal ⁇ 4GlcNAc-structures, animal glycosylations with NeuGc structures.
  • the glycosylation of a recombinant enzyme depends on the glycosylation in the production cell line, these produce partially non-physiological glycan structures.
  • the enzymes are preferably removed from any cell populations aimed for culture or storage or therapeutic use.
  • the presence of enzymes which have affinity with regard to cell surface may otherwise alter the cells as detectable by carbohydrate binding reagents or mass spectrometric or other analysis according to the invention and cause adverse immunological responses.
  • the cell population is cultured or stored in the presence of the modification enzyme to maintain the change in the cell surface structure, when the cell surface structures are recovering from storage especially at temperatures closer physiological or culture temperatures of the cells.
  • the cells are then purified from trace amounts of the modification enzyme before use.
  • the invention is furthermore directed to methods of removal of the modification reagents from cell preparations, preferably the modification reagents are desialylation or resialylation reagents. It is realized that soluble enzymes can be washed from the modified cell populations. Preferably the cell material to be washed is immobilized on a matrix or centrifuged to remove the enzyme, more preferably immobilized on a magnetic bead matrix.
  • the invention is specifically directed to methods for affinity removal of the enzymes.
  • the preferred method includes a step of contacting the modified cells with an affinity matrix binding the enzyme after modification of the cells.
  • the invention is directed to methods of tagging the enzyme to be removed from the cell population.
  • the tagging step is performed before contacting the enzyme with the cells.
  • the tagging group is designed to bind preferably covalently to the enzyme surface, without reduction or without major reduction of the enzyme activity.
  • the invention is further directed to the removal of the tagged enzyme by binding the tag to a matrix, which can be separated from the cells.
  • the matrix comprises at least one matrix material selected from the group: polymers, beads, magnetic beads, or solid phase surface.
  • the invention is furthermore directed to methods of removal of the modification reagents from cells to be depleted of sialic acid and/or resialylated.
  • the preferred modification reagents are desialylation or resialylation reagents.
  • the reagents are tagged to be able to bind the reagents to solid phases comprising specific binder recognizing the tag, the tag binder combination e.g. on microbeads can be removed.
  • Preferred tags include antigens such as peptide FLAG or HA-hemagglutinin peptide tag, or chemical tags such as His-tag or fluoroalkane or biotin.
  • the invention is directed to known specific binders for these, such as specific antibodies for peptides, his-tag binding column for His-TAG, fluoroalkane for hydrogen bond binding of fluoroalkane and avidin or streptavidin for biotin are used.
  • Preferred modification enzymes and enzymes to be tagged include sialidase (neuramini-dases) such as ⁇ 3-, ⁇ 6- and multispecific sialidases and ⁇ 3-, ⁇ 6-sialyltransferases for example from mammalian or bacterial origin and specific for type I and/or type II N-acetyllactosamines, preferably type two N-acetyllactosamines and N-glycans especially biantennary and triantennary N-glycans known in the art.
  • the invention is specifically directed to preferred tagged enzymes as substances.
  • Preferred bacterial fucosyltransferases include enzymes homologous to human ⁇ 3/4-fucosyltransferases, such as Helicobacter pylori fucosyltransferases homologous to enzyme described in Sun H-Y. J. Biol. Chem. (2007) manuscript M601285200, published 24.1.2007 .
  • the preferred specificities of bacterial such as H. pylori fucosyltransferase include reaction with 3'modified lactosamines such as Neu5Ac ⁇ 3Gal ⁇ 4GlcNAc to synthesize sialyl-Lewis x, known to be produced at lest by part of H. pylori strains.
  • the invention is directed to methods of tagging the enzyme to be removed from the cells.
  • a sialidase enzyme or sialyltransferase is linked to tag-molecule, the tagged enzyme is reacted with the cells to be remodelled and the enzyme is removed after the reaction by immobilizing the enzyme by binding to a molecule specifically binding to the tag and the modified cell(s) are removed from the immobilized enzyme by filtering the cells with matrix of a molecule specifically binding to the tag
  • preferred matrices include column used for cell purification or magnetic beads used for purification of components from cell mixtures (see protocols or catalogs of Dynal and Miltenyi companies).
  • the tagging step is preferably performed before contacting the enzyme with the cells.
  • the tagging group is designed to bind preferably covalently to the enzyme surface, without reduction or without major reduction of the enzyme activity.
  • Preferred covalent linkage occurs to amine groups, thiol group or oxidized glycan groups as known from catalogue of Pierce.
  • the invention is further directed to the removal of the tagged enzyme by binding the tag to a matrix, which can be separated from the cells to be modified.
  • Cells proteins are preferably separated from tag-binder immobilized reagents in aqueous media as known in the art of using the tags.
  • the matrix comprises at least one matrix material selected from the group: polymers, beads, magnetic beads, or solid phase surface.
  • the invention is directed to the use for modification of the cells according to the invention, or in a separate embodiment reagents for processes according to the invention, of a human acceptable enzyme, preferably sialidase or sialyltransferase, which is acceptable at least in certain amounts to human beings without causing harmful allergic or immune reactions. It is realized that the human acceptable enzymes may not be needed to be removed from reaction mixtures or less washing steps are needed for desirable level of the removal.
  • the human acceptable enzyme is in preferred embodiment a human glycosyltransferase or glycosidase.
  • the present invention is separately directed to human acceptable enzyme which is a sialyltransferase.
  • the present invention is separately directed to human acceptable enzyme which is a sialidase, the invention is more preferably directed to human sialidase which can remove specific type of sialic acid from cells.
  • the human acceptable enzyme is purified from human material, preferably from human serum, urine or milk.
  • the enzyme is recombinant enzyme corresponding to natural human enzyme. More preferably the enzyme corresponds to human natural enzyme corresponds to natural cell surface or a secreted from of the enzyme, more preferably serum or urine or human milk form of the enzyme. Even more preferably the present invention is directed to human acceptable enzyme which corresponds to a secreted form of a human sialyltransferase or sialidase, more preferably secreted serum/blood form of the human enzyme.
  • the human acceptable enzyme is a controlled reagent with regard to potential harmful glycan structures, preferably NeuGc-structures according to the invention.
  • the recombinant proteins may contain harmful glycosylation structures and inventors revealed that these kinds of structures are also present on recombinant glycosyltransferases, even on secreted (truncated) recombinant glycosyltransferases.
  • Quantitative and qualitative mass spectrometric analysis of modified cells and or reagents The present invention is further directed to the quantitative and qualitative mass spectrometric analysis of modified cells and/or reagents according to the invention.
  • the invention is directed to production of qualitative glycome analysis of the cell and/or the reagents including determining the monosaccharide composition obtained for the materials.
  • the present invention is further directed to quantitative mass spectrometric analysis of the materials according to the invention involving determining the intensities of all or part of the mass spectrometric signals verified to be (reasonably) quantitative with regard to the amount of molecules corresponding to the signals, preferably MALDI-TOF mass spectrometric signals.
  • the invention is further directed to methods, especially research an development methods, such as product development methods, according to the invention for production of reagents or cells as described by the invention involving step of quantitative and/or qualitative glycome analysis, more preferably both quantitative and qualitative analysis.
  • Preferred reagents to be controlled include preferably all reagents derived from or produced in connection with biological material; preferably these include all glycoprotein, protein mixture, serum, and albumin preparations present in the process.
  • the present invention is directed to the control of animal albumins, preferably bovine serum albumin, and human serum albumin preparations for potential contamination by glycan structures.
  • controlled reagents include controlled transferrin and other serum proteins, even more preferably controlled serum proteins are controlled antibody preparations, preferably Fc blocking antibody preparations.
  • the invention is directed to the production of glycan depleted and/or remodelled protein mixtures preferably glycan remodelled human or animal serum, more preferably a serum from an animal used for production of serum products, preferably cell culture serum or antibodies.
  • Preferred serums to be modified includes serum of cow, horse, sheep, goat, rabbit, rat or mouse, more preferably serum of cow, horse, or sheep, even more preferably fetal bovine serum.
  • the glycosylation of the serum is altered by a method based on animals with genetically altered glycan production preferably obtained by a) genetic manipulation of the animal or b) breeding a natural or selecting a natural variant of the production animal to used for serum production, preferably the genetic alteration is directed to tissues producing serum proteins.
  • the present invention is directed under specific embodiment to methods for removal of non-desired carbohydrate structures from living cells.
  • the enzyme proteins are usually antigenic, especially when these are from non-mammalian origin, such as bacteria and/or plants. If the material is not of human origin its glycosylation likely increases the antigenicity of the material. This is particularly the case when the glycosylation has large differences with human glycosylation, preferred examples of largely different glycosylations include: prokaryotic glycosylation, plant type glycosylation, yeast or fungal glycosylation, mammalian/animal glycosylation with Gal ⁇ 3Gal ⁇ 4GlcNAc-structures, animal glycosylation with NeuGc structures.
  • the glycosylation of a recombinant enzyme depends on the glycosylation of the production cell line, these produce partially non-physiological glycan structures in most cases.
  • Glycan depleted biological materials preferably glycoprotein materials
  • Present invention is specifically directed to use biological materials, preferably glycoprotein material, from which harmful structure is removed or reduced in amount.
  • Glycoproteins are major source of bioactive glycans, in some material presence of glycolipids may be also possible and could be handled similarly.
  • the lipid part of glycolipid binds it to the material, released glycan or part of it is water soluble and can be separated.
  • the invention is further directed to glycan depletion methods. In a preferred embodiment the invention is directed to methods including steps of releasing glycan structure and removing released glycan structure.
  • Preferred methods for removal of the released glycan structure include filtration methods.
  • the filtration methods are based on size difference of the released glycan structure and the glycan depleted protein.
  • a preferred method for removal of the released glycans includes precipitation methods, in a preferred embodiment the invention is directed to precipitation of the protein under conditions where the released glycan structure is soluble.
  • the glycan depletion may be combined with a step of inactivation of potential harmful proteins such as lectins or antibodies possibly involved in the process.
  • Some reagents such serum in certain cell culture processes may be heat inactivated.
  • the inactivation may be partial.
  • the partial inactivation is in a preferred embodiment performed by releasing glycans inhibiting the harmful binding proteins to the reagent and further to cell involving process.
  • the depleted glycan and the binding protein inhibiting glycan is the same structure.
  • the released glycans are used when these can not be incorporated to cells to cause further problems in the cell related process.
  • the method of released glycans is not preferred for NeuGc under conditions where it can be incorporated to cells.
  • one or several terminal structures are depleted from a biological material, preferably glycoprotein material.
  • the preferred methods to deplete terminal structures include enzymatic and chemical methods.
  • Preferred enzymatic method is hydrolysis by a glycosidase enzyme or by a trans-glycosylating enzyme capable of removing the terminal structure. Terminal depletion may further include release of several terminal monosaccharide units for example by glycosidase enzymes.
  • Preferred chemical hydrolysis is an acid hydrolysis, preferably a mild acid hydrolysis under conditions not destroying protein structure or from which the protein structure can be restored or renatured.
  • the structure to be depleted is in a preferred embodiment a sialic acid.
  • the sialic acid is preferably released by a sialidase enzyme or by mild acid hydrolysis.
  • the present invention is further directed to internal depletion of glycan material by release of glycans from subterminal linkages by chemical and/or enzymatic methods.
  • Methods to release glycans chemically include base hydrolysis methods such as beta elimination for release of O-linked glycans, hydrazinolysis methods to release O-glycans and N-glycans, oxidative methods such as Smith degradation and ozonolysis (preferred for glycolipids).
  • Preferred enzymatic methods include use of endo-glycosidases such as endoglycosylceramidase for glycolipids, N-glycosidases for N-glycans, and O-glycosidases for O-glycans.
  • endo-glycosidases such as endoglycosylceramidase for glycolipids
  • N-glycosidases for N-glycans
  • O-glycosidases for O-glycans.
  • the present invention is directed to the use of reagents from non-animal sources devoid of potentially harmful reagents.
  • Preferred non-animal glycosylated proteins are proteins from yeasts and fungi and from plants. It is notable that even these materials contain glycans, which may have harmful allergenic activities or which may cause problems in analysis of human type glycans.
  • the invention is further directed to control of the glycosylated reagents from non-animal structures, too.
  • Preferred plant derived proteins include recombinant albumins produced by plant cell culture, more preferably non-glycosylated human serum albumins and bovine serum albumins and recombinant gelatin materials such as collagens produced by plant cell systems.
  • the present invention is specifically directed to the processes according to present invention, when a material containing glycans or harmful glycans according to the present invention is replaced by a reagent, preferably a controlled reagent from non-animal sources.
  • bacterial recombinant proteins are known for lacking expression of glycans.
  • Present invention is directed to control of glycosylation of bacterial protein, as this happens on certain proteins.
  • the present invention is specifically directed to the processes, when a material containing glycans or harmful glycans according to the present invention is replaced by a reagent, preferably a controlled reagent from prokaryotes.
  • the present invention is also specifically directed to the glycan controlled enzyme preparations, especially when produced in a mammalian cell line/cultivation process and controlled with regard to Gal ⁇ 3Gal ⁇ 4GlcNAc-structures, animal glycosylations with NeuGc structures.
  • the preferred enzymes are of human origin, more preferably recombinant enzymes. Most preferably a human serum form of the enzyme is selected and the glycosylation is controlled to be a non-antigenic human-type glycosylation, preferably similar to the glycosylation human natural soluble enzyme.
  • Preferred sialyltransferases includes mammalian, more preferably human ⁇ 3-, and ⁇ 6-sialytransferases, preferably in soluble form.
  • the transferase sialylates N-acetyllactose amines such as ST3GalIII and ST3GalIV or ST6GalI or O-glycans core I such as ST3GalI or ST3GalII and ST3GalIV. It is realized that most effective sialylation is obtained with combination of at least two sialyltransferases such as core I sialylating and N-acetyllactosamine sialylating, e.g. ST3GalIII and ST3GalIV or ST3GalI/II and ST3GalIV.
  • Preferred fucosyltransferases include mammalian, more preferably human ⁇ 3-, and ⁇ 6-fucosyltransferases, preferably in soluble form.
  • the transferase reacts with N-acetyllactosamines such as FTIII, FTIV, FTV, FTVI, FTVII and FTIX more preferably sialyl- ⁇ 3-N-acetyllactosamines, preferably FTIII, FTIV, FTV, FTVI, FTVII, more preferably FTIII, FTV, FTVI, and FTVII, even more preferably FTVI, and FTVII, and most preferably FTVI. It is realized that most effective fucosylation is obtained with combination of at least two fucosyltransferases.
  • the galactosylation reaction is performed in the presence Mg 2+ ions as described in US2005014718 , preferably by mammalian GalT, more preferably natural human GalT, or using exogenous transferase such as Mg 2+ selective ⁇ 4-Galactosyltransferase of Qasba and Ramakrisnan.
  • glycan controlled sialyltransferase or galactosyltransferase is useful to use.
  • the invention is directed to analysis of glycans of non-human expressed glycosyltransferases.
  • the transferases comprise non-human glycosylation
  • the non-human structures are preferably removed by specific glycosidases or modified by chemically e.g. by perjodate oxidation and reduction.
  • the invention is especially directed to use of controlled enzyme substances, preferably galactosyltransferase or sialyltransferase for reaction according to the invention comprising glycan according to Formula Man ⁇ 6(Man ⁇ 3)Man ⁇ 4GlcNAc ⁇ 4(Fuc ⁇ 6)nGlcNAc-N-E wherein E is enzyme protein, N is glycosidic linkage nitrogen in N-glycosylation site (Asn-X-Ser/Thr), and the non-reducing end mannoses may be further modified by (Ne-uNAc ⁇ 3/6)mGal ⁇ 4GlcNAc ⁇ 2, wherein m and n are 0 or 1.
  • controlled enzyme substances preferably galactosyltransferase or sialyltransferase for reaction according to the invention comprising glycan according to Formula Man ⁇ 6(Man ⁇ 3)Man ⁇ 4GlcNAc ⁇ 4(Fuc ⁇ 6)nGlcNAc-N-E wherein E is enzyme protein, N is glycos
  • the enzyme would comprise NeuNGc instead of NeuNAc, this is preferably removed and changed to NeuNAc.
  • the enzymes are non-glycosylated preferably from bacterial production e.g. as described by Qasba US2007258986 (included fully as reference) or N- (Asn-X-Ser/Thr/Cys) and possible O-glycosylation sites of the enzymes are mutated for expression in eukaryotic system.
  • Man ⁇ 4-residue is devoid of Xyl ⁇ 2-modification present in plant cells and reducing end GlcNAc is devoid of Fuc ⁇ 3-structure present in insect or plant cell derived material (e.g. when the enzyme would be produced by insect or plant cell culture).
  • Glycosyltransferase inhibitors for release of glycosyltransferase from cells
  • the present invention is especially directed to use of analogs or derivatives of acceptor saccharides or donor nucleotides for inhibitors of glycosyltransferases for washing the transferase effectively from cells after the reaction.
  • the preferred acceptor analogs include carbohydrates oligosaccharides, monosaccharides and conjugates and analogs thereof capable of binding to substrate site and inhibiting the acceptor binding of the enzyme.
  • the preferred concentrations of the carbohydrates includes concentrations tolerable by the cells from 1 mM to 500 mM, more preferably 5 mM to 250 mM and even more preferably 10-100 mM, higher concentrations are preferred for monosaccharides and method involving solid phase bound binders.
  • Preferred oligosaccharide for sialyltransferase inhibition includes sequences including oligosaccharides and reducing end conjugates includes Gal ⁇ 4Glc, Gal ⁇ 4GlcNAc, Gal ⁇ 3GlcNAc, Gal ⁇ 3GalNAc depending.
  • GalT inhibitors include GlcNAc and conjugates and GlcNAc ⁇ 2Man, GlcNAc ⁇ 6Gal and GlcNAc ⁇ 3Gal.
  • sialyltransferase is released by acceptor disaccharide, more preferably by 5-150 mM acceptor, more preferably by 10-100 mM, even more preferably 10-80 mM, more preferably 10-50 mM for high affinity acceptor and 20- 100 mM, more preferably 40-100 mM, most preferably 50-100 mM for low affinity acceptor.
  • acceptor affinities varies between enzymes, lactose is considered as medium low affinity acceptor for ⁇ 2,3-(N)-Sialyltransferase (Calbiochem) or ST3GalIII and high affinity acceptors have typically acceptor Km values about 10 fold lower.
  • washing removes at least 50% of the cell bound enzyme even more preferably at least, 70%, even more preferably at least 85%, even more preferably at least 90% and most preferably at least 95%.
  • the preferred reducing end structure in conjugates is AR, wherein A is anomeric structure preferably beta for Gal ⁇ 4Glc, Gal ⁇ 4GlcNAc, Gal ⁇ 3GlcNAc, and alfa for Gal ⁇ 3GalNAc and R is organic residue linked glycosidically to the saccharide, and preferably alkyl such as method , ethyl or propyl or ring structure such as a cyclohexyl or aromatic ring structure optionally modified with further functional group.
  • A is anomeric structure preferably beta for Gal ⁇ 4Glc, Gal ⁇ 4GlcNAc, Gal ⁇ 3GlcNAc, and alfa for Gal ⁇ 3GalNAc and R is organic residue linked glycosidically to the saccharide, and preferably alkyl such as method , ethyl or propyl or ring structure such as a cyclohexyl or aromatic ring structure optionally modified with further functional group.
  • Preferred monosaccharides include terminal or two or three terminal monosaccharides of the binding epitope such as Gal, GalNAc, GlcNAc, Man, preferably as anomeric conjugates: as Fuc ⁇ R, Gal ⁇ R, GalNAc ⁇ R, GalNAc ⁇ R GlcNAc ⁇ R, Man ⁇ R.
  • ⁇ 3- or ⁇ 6-sialyltransferase synthesing sialyl Gal ⁇ 4GlcNAc is preferably inhibited by Gal ⁇ 4GlcNAc or lactose.
  • Preferred donor analog includes CMP and derivatives for sialyl-transferases and UDP and derivatives for galactosyltransferases, the analogs preferably interfere also with acceptor binding so that the enzyme is released.
  • the invention is directed to sialyltransferase catalyzed transfer of a natural sialic acid, preferably NeuAc, NeuGc or Neu-O-Ac, from CMP-sialic acid to target cells.
  • the invention provides sialyltransferase catalyzed reaction according to Formula CMP-SA + target cell ⁇ SA-target cell + CMP, preferably CMP-SA + Gal ⁇ 4/3GlcNAc - target cell ⁇ SA ⁇ 3/6Gal ⁇ 4/3GlcNAc - target cell + CMP, wherein SA is a sialic acid, preferably a natural sialic acid, preferably NeuAc, NeuGc or Neu-O-Ac and the reaction is catalysed by a sialyltransferase enzyme preferably by an ⁇ 3-sialyltransferase and the target cell is a cultured stem cell or stem cell or early human blood cell (cord blood cell).
  • Preferred fucosyltransferase reactions synthesis of Lewis a and Lewis x and sialylated variant thereof are: GDP-Fuc + Gal ⁇ 4/3GlcNAc - target cell ⁇ Gal ⁇ /3(Fuc ⁇ 3/4)GlcNAc - target cell + GDP, and/or GDP-Fuc + SA ⁇ 3Gal ⁇ 4/3GlcNAc - target cell ⁇ SA ⁇ 3Gal ⁇ 4/3(Fuc ⁇ 3/4)GlcNAc - target cell + GDP.
  • the reaction is catalysed by a fucosyltransferase enzyme preferably by an ⁇ 3/4-fucosyltransferase.
  • ⁇ 4-fucosyltransferases (Fuc-TIII and -TV) are preferred for synthesis of Lewis a.
  • the novel fucosylated cell populations are preferred for functional studies of the structure.
  • the present invention is directed to glycosyltransferases, especially mammalian sialyl-transferases and fucosyltransferases which are effectively modified on lysine residues by NHS-biotin. This is unexpected as the enzymes contain lysine residues in active site regions, and modification of these would have been likely to reduce substantially or destroy the enzyme activities to non-useful levels.
  • the invention revealed useful but unexpectedly high reagent amounts needed to obtain effectively biotinylated proteins.
  • the invention further revealed that it is possible to obtain unexpectedly high biotinylation level and retaining useful enzyme activity.
  • Preferred protein modified sialyltransferase enzymes or other transferase according to the invention are homologous N-acetyllactosamine sialylating ⁇ 3-sialyltransferases including ST3GalIII, ST3GalIV and ST3GalVI, most preferably ST3GalIII-family enzymes.
  • Preferred specifically protein modified fucosyltransferases include ⁇ 3/4-fucosyltransferases Fuc-TIII, Fuc-TIV, Fuc-TV, Fuc-TVI, Fuc-TVII and Fuc-TIX, more preferably Fuc-TVI and Fuc-TVII and most preferably Fuc-TVI.
  • the invention is directed to biotinylated forms of bacterial glycosyltransferases, especially sialyltransferases and fucosyltransferases.
  • the invention is directed to novel substances according to the formula GE(-Lys)m-[NH-CO-(S)n-tag]q, wherein GE is glycosylation modification enzyme, including glycosyltransferring enzymes, glycocosidases and in separate embodiment also proteases and protease inhibitors; more preferably glycosyltransferase, more preferably sialidase, sialyltransferase or fucosyltransferase according to the invention, Lys is lysine residue, presenting reactive primary amine on side chain, alternatively an amine group may be represented by other structure preferably N-terminus of the protein, S is spacer, n is 0 or 1, indicating either presence or absence of spacer, m is integer from 0 to about 20, varying between enzymes, and indicating the average number of lysines modified, q is integer from 1 to about 20, varying between enzymes, and indicating the number of tag molecules conjugated.
  • GE glycosylation modification enzyme, including glyco
  • GE is sialyltransferase or fucosyltransferase.
  • a low average level biotinylation is performed so that m is at least 0.8, more preferably at least 1.0, even more preferably at least 1.1, but less than 2.
  • Preferred lower range average biotinylation levels for sialyltransferase are about 1.1. (preferably in range 0.8-1.4) and about 1.6 for fucosyltransferase (preferably in range 1.3.-1.9).
  • the lower fucosylation is preferred especially for analysis purposes for observing enzymes on cell surfaces, when quantitative biotinylation of all enzyme proteins is not needed. It is realized that the lower level biotinylated enzymes have properties closer to the native enzyme, e.g. with regard to molecular weight.
  • m is preferably at least 2, even more preferably at least 2,5, even more preferably at least 3.
  • GE is sialyltransferase and m is between 3 and 5, more preferably between 3.2 and 4, even more preferably between 3.25 and 3.8.
  • GE is ⁇ 3-sialytransferase, preferably ST3GalIII and it is modified to amine groups to contain about 3.5, more preferably about 3.4 (3.1-3.7 biotin residues).
  • GE is fucosyltransferase and m is between 3 and 5, more preferably between 3.5 and 4.8 even more preferably between 3.6 and 4.5.
  • ⁇ 3-fucosyltransferase preferably Fuc-TVI is modified to amine groups to contain about 4, more preferably about 4.3 (4.0-4.6 biotin residues).
  • the invention revealed that it is possible to produce highly biotinylated sialyltransferases with useful activity.
  • the high biotinylation level was revealed to include range of biotin residues so that practically each enzyme protein contains a biotin.
  • the highly biotinylated enzymes are especially preferred for removal from the cell modification reactions, practically no enzyme was left.
  • the invention revealed extremely efficient removal of the enzymes from reaction by solid phase conjugated binding reagent for the tag, especially streptavidin coated magnetic particles.
  • the highly biotinylated active enzymes are thus preferred by the invention.
  • the invention is further directed to use of biotinylated enzymes with the reagents revealed to be effective in cell modification such as divalent cations and/or magnesium and/or calsium, supporting factors and non-glycoprotein.
  • highly biotinylated enzyme is used in a medium comprising biotin.
  • the invention is especially directed to removal of the highly biotinylated enzymes even from biotin containing solutions, at least when the biotin concentration is not substantially higher than that in ⁇ -MEM, and optionally 1-2 washing steps are included to remove excess biotin, before the removal of the enzyme.
  • the invention is directed to the use of the supporting factors according to the invention and excluding biotin or using another tag such as fluorocarbon or peptide tag not included in the reaction mixtures.
  • the invention is further in a preferred embodiment directed to combined fucosyltransferase and sialyltransferase reaction with a broad specificity, sialyltransferase sialylating ⁇ 3/4-fucosylated terminal oligosaccharide sequences such as Lewis x and/or Lewis a, preferably at least Lewis x, and an ⁇ 3/4-fucosyltransferase reacting with both sialylated and non-sialylated acceptors.
  • sialyltransferase and fucosyltransferase reactions for synthesis of sialyl-Lewis x and/or sialyl-Lewis a Gal ⁇ 4/3GlcNAc (Sialyltransferase and fucosyltransferase) ⁇ NeuNAc ⁇ 3Gal ⁇ 4/3(Fuc ⁇ 3/4)GlcNAc.
  • broad specificity microbial enzyme is used for sialylation of Lewis x and optionally also LacNAcs on cell surface Gal ⁇ 4(Fuc ⁇ 3)GlcNAc (broad specificity Sialyltransferase) ⁇ NeuNAc ⁇ 3Gal ⁇ 4(Fuc ⁇ 3)GlcNAc
  • the present invention is directed to the use of the specific enzyme for or in context of modification of the stem cells wherein the enzyme is covalently conjugated to a tag.
  • the conjugation according to the invention may be performed non-specifically, e.g. by biotinylation one or several of multiple amines on the cell surface, or specifically.
  • the specific conjugation aims for conjugation from protein regions, which does not disturb the binding of the binding site of the enzyme to its ligand glycan and/or donor nucleotide binding site of a glycosyltransferase to be modified on the cell surface glycans of stem cells according to the invention.
  • Preferred specific conjugation methods include chemical conjugation from specific amino acid residues from the surface of the enzyme protein/peptide.
  • specific amino acid residue such as cysteine is cloned to the site of conjugation and the conjugation is performed from the cysteine.
  • N-terminal cysteine is oxidized by periodic acid and conjugated to aldehyde reactive reagents such as amino-oxymethyl hydroxylamine or hydrazine structures
  • further preferred chemistries include "Click" chemistry marketed by Invitrogen and amino acid specific coupling reagents marketed by Pierce and Molecular probes.
  • a preferred specific conjugation occurs from protein linked carbohydrate such as O- or N-glycan of the enzyme, preferably when the glycan is not close to the binding site of enzyme substrates or longer spacer is used.
  • Preferred glycan conjugation occurs through a reactive chemoselective ligation group R1 of the glycans, wherein the chemical group can be specifically conjugated to second chemoselective ligation group R2 without major or binding destructive changes to the protein part of the enzyme.
  • Chemoselective ligation groups reacting with aldehydes and/or ketones include as amino-oxy- methyl hydroxylamine or hydrazine structures.
  • a preferred R1-group is a carbonyl such as an aldehyde or a ketone chemically synthesized on the surface of the protein.
  • Other preferred chemoselective groups include maleimide and thiol; and "Click"-reagents (marketed by Invitrogen) including azide and reactive group to it.
  • Preferred synthesis steps include
  • oxidative enzymes or periodic acid are known in the art having been described in patent application directed conjugating HES-polysaccharide to recombinant protein by Kabi-Frensenius ( WO2005 EP02637 , WO2004 EP08821 , WO2004 EP08820 , WO2003 EP08829 , WO2003 EP08858 , WO2005092391 , WO2005014024 included fully as reference) and a German research institute.
  • Preferred methods for the transferring the terminal monosaccharide reside includes use of mutant galactosyltransferase as described in patent application by part of the inventors US2005014718 (included fully as reference) or by Qasba and Ramakrishman and colleagues US2007258986 (included fully as reference) or by using method described in glycopegylation patenting of Neose ( US2004132640 , included fully as reference).
  • the enzyme is, specifically or non-specifically conjugated to a tag, referred as T, specifically recognizable by a ligand L
  • tags includes such as biotin binding ligand (strept)avidin or a fluorocarbonyl binding to another fluorocarbonyl or peptide/antigen and specific antibody for the peptide/antigen
  • the preferred conjugate structures are according to the Formula CONJ B-(G-)mR1-R2-(S1-)nT, Wherein B is the enzyme, G is glycan (when the enzyme is glycan conjugated), R1 and R2 are chemoselective ligation groups, T is a tag, preferably biotin; S1 is an optional spacer group, preferably C1-C10 alkyls, m and n are integers either 0 or 1, independently.
  • a preferred method of the tag conjugate involves following steps:
  • the matrix comprising the ligand may be solid phase or affinity matrix or polymer or other matrix useful for removal of the enzyme.
  • the matrix may be used in form of magnetic particles, column, surface of tubing or vessel, soluble or insoluble preferably water miscible polymer.
  • the tagged enzyme is used together with non-tagged enzyme in order to establish the level of non-tagged enzyme with same or very similar cell binding properties in a cell preparation, preferably aimed for therapeutic use, and removal of the tagged enzyme.
  • the invention further revealed novel glycan conjugated enzymes. It is realized that the glycan conjugation surprisingly maintained the enzyme activity and allows effective reactions.
  • the invention is further directed multiply Tag conjugated glycans preferably being on average about 3-15 tags, more preferably 4-12, most preferably 4-10 tags per enzyme.
  • the invention is especially directed to multiply conjugated tags on enzymes and/inhibitors according to the invention according to the Formula Formula CONJm B-(G-)m[R1-R2-(S1-)nT]q
  • B is the enzyme
  • SOL is solid phase or affinity matrix or polymer or other matrix useful for removal of the enzyme
  • G is glycan (when the enzyme is glycan conjugated)
  • R1 and R2 are chemoselective ligation groups
  • T is tag, preferably biotin
  • L is specifically binding ligand for the tag
  • S1 is an optional spacer group, preferably C1-C10 alkyls
  • m is integer from 1-50, indicating number of glycan epitopes to be modified
  • n is integer being either 0 or 1, independently
  • q is integer from 1 to about 20, varying between enzymes, and indicating the number of tag molecules conjugated.
  • the invention is further directed to multivalent conjugates.
  • the enzyme is removed by using the tag following complex structure is preferably formed according to Formula COMPm B-(G-)m[R1-R2-(S1-)nT-L-(S2)s-SOL]q, q is integer from 1 to about 20, varying between enzymes, and indicating the number of tag molecules conjugated, other variables are as described for Formula COMP.
  • the bacterial enzymes are especially useful for reactions according to the invention due to high specific activity and even higher useful specificity ranges.
  • the invention revealed high activity of the enzymes under preferred cell modification conditions containing physiological salt levels.
  • the reaction conditions in previous publications have included non-useful reaction conditions for cell modification e.g. 0.5 M salt for Photobacterium enzyme ( Tsukamoto H. et al., J. Biol. Chem 2007, 282, 29794-801 ).
  • the novel preferred microbial sialyltransferases, including Myxovirus and Photobacterium enzymes for cell modification are shown in Figure 4b .
  • the preferred bacterial enzymes includes ⁇ 2,3-sialyltransferase from Photobacterium phosphorium and ⁇ 2,6-sialyltransferase from Photobacterium damselae.
  • the invention is directed to use of sialyltransferase which can sialylate (i) both fucosylated and non-fucosylated type II N-acetyllactosamines Gal ⁇ 4GlcNAc and Gal ⁇ 4(Fuc ⁇ 3)GlcNAc and (ii) optionally terminal type I N-acetyllactosamines Gal ⁇ 3GlcNAc and Gal ⁇ 3(Fuc ⁇ 3)GlcNAc and (iii) even more preferably also Gal ⁇ 3GalNAc ⁇ sequences in O-glycans and/or Gal ⁇ 3GalNAc ⁇ in glycolipids.
  • a preferred sialyltransferase for sialylation for all the preferred acceptor types including type I and type II lactosamines, and glycolipid Gal ⁇ 3GalNAc ⁇ and fucosylated acceptors Gal ⁇ 4(Fuc ⁇ 3)GlcNAc is a viral sialyltransferase from myxoma virus v-ST3GalI ( Sujino et al. Glycobiology 2000 10 (3) 313-20 ). It is realized that the broad specificity sialyltransferases sialylate cells most effectively to multiple acceptor sites.
  • the sulfated N-acetyllactosamines of human mesenchymal cells preferably mesenchymal stem cells contain sulfate on both Gal and GlcNAc residues of the N-acetyllactosamines (LacNAcs).
  • Preferred reactions with (SE6)GlcNAc containing structures includes fucosylation, sialylation or fucosylation and sialylation to synthesize a) 6-sulfo-sialyl-LacNAc, b) 6-sulfo-Lewis x and c) 6-sulfo-sialyl-Lewis x: according to formulas:
  • Preferred reactions with (SE3)Gal containing structures include fucosylation, a) 3'sulfo-fucosyl-LacNAc according to the reaction formula:
  • Preferred reactions with (SE3)Gal ⁇ 4(SE6)GlcNAc containing structures includes fucosylation, a) 3'-,6-sulfo-fucosyl-LacNAc according to the reaction:
  • the invention is further directed to spontaneous fucosylation reactions to synthesize sialylated and/or fucosylated sulfo-N-acetyllactosamines.
  • Preferred reagents for the recognition of acceptor structures, preferably on non-modified cells according to the invention include sulfo-lactosamine structure recognizing antibodies, preferably 3'sulfo-LacNAc and 6-sulfo LacNAc, 3'-,6-sulfo-LacNAc specific antibodies and for the recognition of product structures antibodies for the corresponding fucosylated and/or sialylated structures.
  • sulfo-lactosamine structure recognizing antibodies preferably 3'sulfo-LacNAc and 6-sulfo LacNAc, 3'-,6-sulfo-LacNAc specific antibodies and for the recognition of product structures antibodies for the corresponding fucosylated and/or sialylated structures.
  • Examples of antibodies are in Table 3, in case specific antibody is not available combinations of the antibodies can be used or combinations of antibody and glycosidase analysis.
  • BM-MSC Bone marrow-derived mesenchymal stem cells
  • HSA human serum albumin
  • Immunophenotypic markers for MSC binding of conjugated lectins and glycoform-specific antibodies for Lex and sLex glycan epitopes were analyzed by flow cytometry directly after trypsinization.
  • the detached cells were centrifuged 300 x g for 5 min, the supernatant was completely removed and 1x106 cells were resuspended in 300 ⁇ l enzyme reaction buffer composed of Minimum Essential Medium (MEM) ⁇ medium supplemented with 0.5% HSA (reaction buffer control).
  • MEM Minimum Essential Medium
  • Glycosylation enzymes and sugar donors were added into the reaction buffer (Minimum Essential Medium (MEM) ⁇ medium supplemented with 0.5 % HSA) in the following concentrations: 50 or 100 mU rat recombinant ( Spodoptera frugiperda ) ⁇ 2,3-(N)-Sialyltransferase III (SAT) (Calbiochem) and 1 mg CMP-NeuAc, 15 or 30 mU human recombinant ( Spodoptera frugiperda ) ⁇ 1,3-Fucosyltransferase VI (FUT) (Calbiochem) + 1 mg GDP-fucose, both SAT and FUT or 30 or 60 mU Sialidase (Neuraminidase) C.
  • the original enzyme buffers were exchanged to the above mentioned reaction buffer or PBS (for Sialidase C.perfringens ) by dialysis.
  • the BM-MSC cell suspensions were incubated in 24-well cell culture vessels for maximally 2 hours in +37°C cell incubator, except in the double SAT+FUT reactions, wherein FUT and GDP-Fucose were added after one hour prior incubation with SAT.
  • the reactions were resuspended by mechanical pipetting using ART 1000E filter tips (Molecular BioProducts) with M1000 micropipette (Biohit) every 20 minutes during the incubation. Parallel reactions with cells only in the reaction buffer without the enzymes were always included in each experiment. The reactions were collected after 2 hours and cell viability and the number of cells were determined with trypan blue exclusion.
  • FACS analysis For immunophenotypic characterization of MSC by flow cytometry (FAC-SAria, Becton Dickinson), antibodies against the following molecules were used (table x): CD90, CD73, CD105, HLA-ABC, CD34, CD45, CD14, CD19 and HLA-DR.
  • Cell surface lectin binding profile of the BM-MSCs was studied with FITC- or biotin- conjugated MAA, SNA, RCA and ECA lectins before and after the enzymatic modifications of cell glycans.
  • FITC-conjugated anti-streptavidin secondary antibody staining was done when biotin-conjugated lectins were used.
  • Alexa 488-conjugated anti- Lex/sLex glycoform antibodies were also used for FACS analysis: CD 15 (TG-1) Lex, PSGL-1 (core 2 O-glycan) sLex and sCD15 (sLex). 1 x 10 5 BM-MSCs were labelled in Ca 2+ -free PBS supplemented with 2 mM EDTA and 1% BSA. Analysis was performed using the FACSDiva software (Beckton Dickinson).
  • BM-MSC viability and immunophenotypic markers after in vitro glycomodification Viability of the BM-MSCs never fell below 98% and their cell surface expression of CD90, CD73, CD105, HLA-ABC, CD34, CD45, CD14, CD19 and HLA-DR remained unaltered after the ⁇ 2,3-sialylation (SAT) or ⁇ 1,3-fucosylation (FUT) reactions carried out in the reaction conditions described above (data not shown).
  • SAT ⁇ 2,3-sialylation
  • FUT ⁇ 1,3-fucosylation
  • the ⁇ 1,3-fucosylation reaction was very efficient and > 90 % of the cells expressed the sLex/CSLEX1 epitope in the reaction composition and time chosen ( Figure 1 , sLex/CSLEX1 antibody). Also the core-2 sLex antibody stained >90 % of the cells after the ⁇ 1,3-fucosylation reaction. A proceeding 1 h ⁇ 2,3-sialylation inhibited the fucosylation reaction ( Figure 1 ).
  • BM-MSCs in 70% confluency were detached with 0.25% trypsin/1 mM EDTA in Ca 2+ /Mg 2+ -free PBS (Invitrogen) for 3 minutes. Trypsinization was inhibited by adding excess ⁇ -MEM+10% human serum albumin HSA (Albumin SPR, Sanquin, the Netherlands). The cell suspension was centrifuged, supernatant removed and 1.75x10 6 cells were resuspended in either 300 ⁇ l Minimum Essential Medium (MEM) ⁇ medium supplemented with 0.5 % HSA or Ca 2+ /Mg 2+ -free Hanks' Balanced Salt Solution (HBSS) supplemented with 0.1% HSA.
  • MEM Minimum Essential Medium
  • HBSS Hanks' Balanced Salt Solution
  • the BM-MSC cell suspensions were incubated in 24-well cell culture vessels for 2 hours in +37°C cell incubator. In a part of the culture wells, the cells were resuspended by mechanical pipetting through ART 1000E filter tips (Molecular BioProducts) attached to M1000 micropipette (Biohit) every 20 minutes during the incubation. Representative phase contrast microscope pictures of cells in suspension were taken at the beginning of the experiment and after 1 h and 2 h. The cells in suspension were collected at the end of the experiment and cell viability and the number of cells were determined by the trypan blue exclusion.
  • BM-MSCs in suspension exhibited a heterogeneous cell population with cells of different sizes directly after trypsinization (time point 0' ). After 1-2 hour incubation a change in cell morphology could be observed between cells in the ⁇ -MEM reaction buffer and Ca 2+ /Mg 2+ -free HBSS.
  • the cell suspension in Ca 2+ /Mg 2+ -free HBSS contained more large, granular cells and small cells with bleb-like structures on their plasma membrane.
  • the cells suspended in the Ca 2 +- and Mg 2+ -containing ⁇ -MEM reaction buffer contained more cells characterized by a round and clear, mononuclear cell-like morphology and intact plasma membranes (in Figure 2 , compare cells marked with arrows in panels).
  • the cells aggregated more to each others in the ⁇ -MEM reaction buffer, but the aggregation could be inhibited by mechanical resuspension every 20 min. None of the reaction conditions could completely inhibit the cells from adhering to the cell culture vessel bottom as indicated in the total number of cells in suspension after 2 h incubation (Table 2).
  • the adherence to cell culture bottom could also be inhibited by the sequential resuspension.
  • There were a higher number of viable cells in the suspension phase when the cells were incubated in the ⁇ -MEM + 0.5 % HSA reaction buffer than when incubated in the Ca 2+ /Mg 2+ -free HBSS (Table 2).
  • BM-MSC in 70% confluency were detached with either 0.25% trypsin/1 mM EDTA in Ca 2+ /Mg 2+ -free PBS (Invitrogen) or 0.05% trypsin/0.5 mM EDTA in Ca 2+ /Mg 2+ -free HBSS for 3 minutes. Trypsinization was inhibited by adding excess of either ⁇ -MEM+10% fetal calf serum (FCS) (Invitrogen) or ⁇ -MEM+10% human serum albumin (HSA) (Albumin SPR, Sanquin, the Netherlands). Cell viability and the number of cells were determined with trypan blue exclusion and representative phase contrast microscope pictures were taken with lOx objective.
  • FCS fetal calf serum
  • HSA human serum albumin
  • Glycosylation of commercial sialyl- or fucosyltransferase (Calbiochem CA) enzyme produced in insect cells is controlled by releasing the glycans, purifying the glycans and MALDI-TOF mass spectrometry (WO publication by the inventors, filed 11.7.2005).
  • Potentially allergenic insect type glycans are observed and released by exoglycosidase enzymes as described for the insect glycans such as ⁇ -mannosidase, ⁇ -mannosidase ⁇ 3- or ⁇ 3/ ⁇ 6-fucosidases and hexosaminidase (such as Jack bean hexosaminidase), (WO publication by the inventors, filed 11.7.2005).
  • glycosylated glycosyltransferase enzymes can contaminate cells in modification reactions. For example, when cells were incubated with recombinant fucosyltransferase or sialyltransferase enzymes produced in S. frugiperda cells, N-glycosidase and mass spectrometric analysis of cellular and/or cell-associated glycoproteins resulted in detection of an abundant neutral N-glycan signal at m/z 1079, corresponding to [M + Na] + ion of Hex3HexNAc2dHex1 glycan component (calc. m/z 1079.38).
  • this glycan signal was more abundant than or at least comparable to the cells' own glycan signals, indicating that insect-derived glycoconjugates are a very potent contaminant associated with recombinant glycan-modified enzymes produced in insect cells. Moreover, this glycan contamination persisted even after washing of the cells, indicating that the insecttype glycoconjugate corresponding to or associated with the glycosyltransferase enzymes has affinity towards cells or has tendency to resist washing from cells.
  • N-glycan structures e.g. Man ⁇ 3(Man ⁇ 6)Man ⁇ 4GlcNAc(Fuc ⁇ 3/6)GlcNAc( ⁇ -N-Asn), have been described previously from glycoproteins produced in S.
  • frugiperda cells (Staudacher et al., 1992; Kretzchmar et al., 1994; Kubelka et al., 1994; Altmann et al., 1999).
  • glycan-modifying enzymes must be carefully selected for modification of human cells, especially for clinical use, not to contain immunogenic glycan epitopes, non-human glycan structures, and/or other glycan structures potentially having unwanted biological effects or in a preferred embodiment the glycan structures are removed or degraded to non-harmful ones.
  • the SAT and FUT enzymes were recombinant rat a2,3-N-sialyltransferase and recombinant ⁇ 1,3-fucosyltransferase VI, respectively, expressed in S. frugiperda insect cells (Calbiochem).
  • the enzymes were biotinylated with N-hydroxysuccinimide-activated sulfo-biotin with aminocaproate spacer (sulfo-NHS-LC-biotin; Pierce).
  • 5 ⁇ g of enzyme was incubated with either 0.65 nmol (about 5 times molar excess) or 6.5 nmol (about 50 times molar excess) biotinylation reagent in 50 ⁇ l of 50 mM HEPES buffer pH 8 at +4°C for 2 hours.
  • the reactions were stopped by adding a molar excess of Tris-HCl buffer pH 7.5.
  • glycerol-containing enzyme For periodate oxidation, the commercial glycerol-containing enzyme preparates were transferred into phosphate buffered saline solution with gel filtration chromatography in NAP-5 columns (Amersham-Pharmacia/GE Healthcare). Periodate oxidation of glycan chains was performed for 5 ⁇ g of either SAT or FUT in 40 ⁇ l of phosphate buffered saline (PBS) containing 8 mM NaIO 4 , at +4°C in the dark for 2-3 hours.
  • PBS phosphate buffered saline
  • Biotin conjugation to oxidized glycan chains was performed by adding 6.5 nmol (about 100 times excess) of biotin-amido-hexanoic acid hydrazide (BAAH; Pierce) to the glycan-oxidized enzymes and incubating at +4°C for 2-16 hours. The enzymes were isolated for subsequent use.
  • BAAH biotin-amido-hexanoic acid hydrazide
  • a typical radiochemical SAT assay 0.4 mU/ ⁇ l enzyme was incubated with 1 mM CMP-[C-14]Neu5Ac and 0.5 mM acceptor glycoprotein (asialofetuin from Sigma-Aldrich) in 50 mM MOPS buffer pH 7.4 at +37°C. Aliquots from various time points were collected. Protein fraction was separated from the donor substrate by reversed phase solid-phase extraction and measured by liquid scintillation counting according to standard procedures. The amounts of radioactivity transferred to protein were compared. FUT activity was measured similarily using 0.04 mU/ ⁇ l FUT, radiolabeled GDP-Fuc donor, and additionally 20 mM MnCl 2 .
  • 0.2 mU/ ⁇ l enzyme was incubated with 8 mM CMP-Neu5Ac and 4 mM LacNAc acceptor glycan (lacto-N-neotetraose) in 50 mM MOPS buffer pH 7.4 at +37°C, optionally containing 0.2 mg/ml albumin. Aliquots from various time points were collected. FUT activity was measured similarily using 0.05 mU/ ⁇ l FUT, 8 mM GDP-Fuc donor, 4 mM acceptor, optionally with 20 mM MnCl2, and preferentially without albumin. The reaction level was analyzed by MALDI-TOF mass spectrometry using standard procedures, and relative signal intensities (correlating with corresponding relative molar amounts in the sample) of acceptor and product glycans were compared.
  • SAT and FUT were optionally changed into cell-optimized solutions, preferentially ⁇ -MEM (Gibco/Invitrogen) and optionally using filtration concentration or dialysis.
  • ⁇ -MEM Gibco/Invitrogen
  • HSA human serum albumin
  • Conjugation level was analyzed by comparing molecular mass of non-conjugated and conjugated proteins in MALDI-TOF mass spectrometry using standard procedures.
  • Biotinylated enzymes were removed from solution by contacting them with affinity matrix, preferentially with streptavidin-coated magnetic particles (Dynal). Removal of enzyme was visualized by SDS-PAGE and blotting with streptavidin-peroxidase conjugate and color reaction according to standard procedures.
  • the glycan-conjugated SAT showed similar activity as non-conjugated SAT when analyzed by the mass spectrometric assay.
  • Mass spectrometric analysis of glycan conjugated sialyltransferase indicated high conjugation to glycans being about 5-10 biotins per enzyme.
  • biotin-conjugated proteins could be readily removed from solution by streptavidin-coated magnetic particles, as visualized for protein-biotin-conjugated SAT in Figure 6 .
  • the SAT and FUT enzyme preparations in ⁇ -MEM and in ⁇ -MEM supplemented with HSA were active as measured by the mass spectrometric assay. Further, they showed good stability. For example, the HSA-containing SAT preparation in ⁇ -MEM was stable at +4°C for over 16 days (in which time over 75% of original activity was retained as measured by the mass spectrometric assay). Further, FUT in HSA-containing ⁇ -MEM was active without adding Mn 2+ or other divalent cations to the standard ⁇ -MEM.
  • the SAT enzymes were recombinant ⁇ 2,3-sialyltransferase (JT-ISH-224 and JT-ISH-467) from Photobacterium phosphorium and ⁇ 2,6-sialyltransferase (JT160) from Photobacterium damselae expressed in E. coli (Japan Tobacco Inc., Shizuoka, Japan).
  • 0.2 mU/ ⁇ l enzyme was incubated with 8 mM CMP-Neu5Ac and 4 mM LacNAc acceptor glycan (lacto-N-neotetraose) in 20 mM Tris-HCl buffer pH 7.5 at +37°C, containing 0.2 mg/ml albumin and sodium chloride concentration of 150 mM (up to 0.5M was adviced by the manufacturer). Aliquots were collected from several time points.
  • reaction level was analyzed by MALDI-TOF mass spectrometry using standard procedures, and relative signal intensities (correlating with corresponding relative molar amounts in the sample) of acceptor and product glycans were compared, and the enzymes were highly active towards the acceptor.
  • SATs were optionally changed into cell-optimized solutions, preferentially ⁇ -MEM (Gibco/Invitrogen) and optionally using filtration concentration or dialysis.
  • Human serum albumin HSA; Finnish Red Cross Blood Service
  • the salt concentration was lowered to physiological salt contained within the ⁇ -MEM.
  • the enzyme was highly active when analyzed by MALDI-TOF mass spectrometry with lacto-N-tetraose acceptor as described above.
  • the activity of the enzymes was measured relative to equivalent nominal activities according to manufacturer's specifications.
  • the bacterial enzyme was over 10 times more active than the recombinant mammal ⁇ 2,3-N-sialyltransferase produced in insect cells by Calbiochem.
  • Neuraminidase or sialyltransferase is biotinylated as described in catalog of Pierce. Biotinylated neuraminidase or sialyltransferase enzyme is incubated with cells to modify the glycosylation of the target cells as described in the invention. The enzyme is removed by (strept)avidin magnetic beads (e.g. Miltenyi or Dynal) optionally with presence of neura-minic acid and or sialyltransferase acceptor (N-acetyllactosamine or lactose).
  • strept avidin magnetic beads
  • neura-minic acid and or sialyltransferase acceptor N-acetyllactosamine or lactose
  • BM-MSCs Bone marrow-derived mesenchymal stem cells
  • ⁇ 2,3-Sialyltransferase enzymatic modification Cell culture media was collected for further analysis before the detachment.
  • BM-MSCs at 70-80% confluency were detached with PBS + 2mM Na-EDTA (Versene) for 30 min at +37°C.
  • the number of detached cells were calculated in Bürker chamber and control (0-) cells were washed four times in cold Ca 2+ -free PBS and frozen as cell pellets at -70°C for mass spectrometric analysis. All centrifugation steps were performed at 300 x g for 5 min.
  • the enzymatic reactions were performed in 24-well cell culture plates in a humidified 5% CO2 atmosphere at +37°C for either 2 or 4 hours.
  • the reactions were controlled for attachment to the cell culture dish by suspending the cells every 30 min during the incubations. Control reactions were performed simultaneously with cells in reaction buffer only for 2 or 4 hours.
  • the enzymatic reactions were stopped by adding excess (2 ml) of cold Ca 2+ -free PBS or Ca 2+ -free PBS supplemented with 75 mM lactose to the reactions.
  • Cell viability was determined by trypan blue staining and microscopic analysis in a Bürker chamber. The cells were centrifuged at 300 x g for 5 min and washing was repeated additionally 3 times. After the last wash the cells were divided in two and half the cells were pelleted by centrifugation and frozen at -70°C for further mass spectrometric N-glycan analysis and half the cells were used subsequently in flow cytometric analysis.
  • the MALDI-TOF mass spectrometric analysis was performed for N-glycosidase F liberated N-glycans essentially as described (Hemmoranta H. et al., 2007. Exp. Hematol.).
  • Cellular glycan modification with sialyltransferase In the reaction, cell surface terminal N-acetyllactosamine (LN) units were sialylated as demonstrated by N-glycan structural analyses as follows. Reaction efficiency and level of terminal LN modification was followed by MALDI-TOF mass spectrometric profiling of the neutral N-glycan fraction.
  • LN N-acetyllactosamine
  • Three glycan signals were used as indicators of the reaction efficiency, namely at m/z 1622 (corresponding to hybrid-type N-glycan Hex6HexNAc3 Na-adduct signal, with documented one terminal LN unit), m/z 1663 (corresponding to complex-type N-glycan Hex5HexNAc4 Na-adduct signal, with documented two terminal LN units), and m/z 2028 (corresponding to complex-type N-glycan Hex6HexNAc5 Na-adduct signal, with documented three terminal LN units). These three indicator signals were good indicators for the overall sialylation level change.
  • reaction level is calculated by the equation: 100 % - 100 % * I ⁇ 1622 + I ⁇ 1663 + I ⁇ 2028 ⁇ a / I ⁇ 1622 + I ⁇ 1663 + I ⁇ 2028 ⁇ b
  • Ix is relative proportion of glycan signal x (% of total glycan profile)
  • "a” indicates signals after the enzyme reaction
  • "b” indicates those in the control reaction.
  • the glycan signal at m/z 1257 (control, corresponding to Na-adduct of Hex5HexNAc2 high-mannose type N-glycan) stayed between 5.7% - 6.8% in all conditions, showing that modification was specific.
  • glycoprotein components containing a LN group when added to the medium, were efficient substrates of the enzymatic modification, therefore competing with the cellular modification if added to the reaction solution.
  • m/z 1079 Contamination with insect derived enzyme and its removal: m/z 1079, corresponding to sodium adduct ion of Hex3HexNAc2dHex1, a paucimannosidic insect N-glycan / low-mannose type human N-glycan.
  • Mammalian glycosyltransferase e.g. ⁇ 4-galactosyltransferase, bovine GalT1
  • ⁇ -sialidase e.g. ⁇ 4-galactosyltransferase, bovine GalT1
  • Ketone modified Gal is transferred from ketone modified Gal-UDP to the terminal monosaccharide GlcNAc-residue by mutant galactosyltransferase as described in patent application by part of the inventors US2005014718 (included fully as reference) or by Qasba and Ramakrishman and colleagues US2007258986 (included fully as reference) or by using methods described in glycopegylation patenting of Neose ( US2004132640 , included fully as reference).
  • the ketone is reacted with excess of amino-oxy-biotin (or hydrazide-biotin).
  • Umbilical cord blood Human term umbilical cord blood (UCB) units were collected after delivery with informed consent of the mothers and the UCB was processed within 24 hours of the collection.
  • the mononuclear cells (MNCs) were isolated from each UCB unit diluting the UCB 1:1 with phosphate-buffered saline (PBS) followed by Ficoll-Paque Plus (Amersham Biosciences, Uppsala, Sweden) density gradient centrifugation (400 g / 40 min). The mononuclear cell fragment was collected from the gradient and washed twice with PBS.
  • PBS phosphate-buffered saline
  • Ficoll-Paque Plus Amersham Biosciences, Uppsala, Sweden
  • CD45/Glycophorin A (GlyA) negative cell selection was performed using immunolabeled magnetic beads (Miltenyi Biotec). MNCs were incubated simultaneously with both CD45 and GlyA magnetic microbeads for 30 minutes and negatively selected using LD columns following the manufacturer's instructions (Miltenyi Biotec). Both CD45/GlyA negative elution fraction and positive fraction were collected, suspended in culture media and counted. CD45/GlyA positive cells were plated on fibronectin (FN) coated six-well plates at the density of 1x10 6 /cm 2 .
  • FN fibronectin
  • CD45/GlyA negative cells were plated on FN coated 96-well plates (Nunc) about 1x10 4 cells/well. Most of the non-adherent cells were removed as the medium was replaced next day. The rest of the non-adherent cells were removed during subsequent twice weekly medium replacements.
  • the cells were initially cultured in media consisting of 56% DMEM low glucose (DMEM-LG, Gibco, http://www.invitrogen.com ) 40% MCDB-201 (Sigma-Aldrich) 2% fetal calf serum (FCS), 1x penicillin-streptomycin (both form Gibco), 1x ITS liquid media supplement (insulin-transferrin-selenium), 1x linoleic acid-BSA, 5x10-8 M dexamethasone, 0.1 mM L-ascorbic acid-2-phosphate (all three from Sigma-Aldrich), 10 nM PDGF (R&D systems, http://www.RnDSystems.com ) and 10 nM EGF (Sigma-Aldrich). In later passages (after passage 7) the cells were also cultured in the same proliferation medium except the FCS concentration was increased to 10%.
  • FCS fetal calf serum
  • Plates were screened for colonies and when the cells in the colonies were 80-90% confluent the cells were subcultured. At the first passages when the cell number was still low the cells were detached with minimal amount of trypsin/EDTA (0.25%/1mM, Gibco) at room temperature and trypsin was inhibited with FCS. Cells were flushed with serum free culture medium and suspended in normal culture medium adjusting the serum concentration to 2 %. The cells were plated about 2000-3000/ cm2. In later passages the cells were detached with trypsin/EDTA from defined area at defined time points, counted with hemato-cytometer and replated at density of 2000-3000 cells/cm 2 .
  • Bone marrow-derived stem cells Bone marrow-derived stem cells.
  • Bone marrow (BM) -derived MSCs were obtained as described by Leskelä et al. (2003). Briefly, bone marrow obtained during orthopedic surgery was cultured in Minimum Essential Alpha-Medium ( ⁇ -MEM), supplemented with 20 mM HEPES, 10% FCS, 1x penicillin-streptomycin and 2 mM L-glutamine (all from Gibco). After a cell attachment period of 2 days the cells were washed with Ca 2+ and Mg 2+ free PBS (Gibco), subcultured further by plating the cells at a density of 2000-3000 cells/cm 2 in the same media and removing half of the media and replacing it with fresh media twice a week until near confluence.
  • ⁇ -MEM Minimum Essential Alpha-Medium
  • FITC- and PE-conjugated isotypic controls were used.
  • Unconjugated antibodies against CD90 and HLA-DR both from BD Biosciences were used for indirect labeling.
  • FITC-conjugated goat anti-mouse IgG antibody Sigma-Aldrich was used as a secondary antibody.
  • the UBC derived cells were negative for the hematopoietic markers CD34, CD45, CD 14 and CD133.
  • BM-derived cells showed to have similar phenotype. They were negative for CD14, CD34, CD45 and HLA-DR and positive for CD13, CD29, CD44, CD90, CD105 and HLA-ABC.
  • the cells were collected with 1.5 ml of PBS, transferred from 50 ml tube into 1.5 ml collection tube and centrifuged for 7 minutes at 5400 rpm. The supernatant was aspirated and washing repeated one more time. Cell pellet was stored at -70°C and used for glycome analysis.
  • the cells can be used for in vivo imaging trials and in animal models such as PET imagining e.g. as described in Min JJ et al. (2006) Ann Nucl Med 20,(3) 165-70 or Kang WJ et al. (J Nucl Med 47, 1295-1301 ).
  • BM-MSCs line 168 Bone marrow-derived mesenchymal stem cells (BM-MSCs) line 168 were obtained as described by Leskelä et al. (2003). After initial culture establishment, BM-MSCs ( ⁇ passage 10) were cultured in a humidified 5 % CO 2 atmosphere at +37°C in Minimum Essential Alpha-Medium ( ⁇ -MEM) (Invitrogen) supplemented with 10% FCS, 20mM Hepes, 10ml/l penicillin/streptomycin and 2mM L-glutamine.
  • ⁇ -MEM Minimum Essential Alpha-Medium
  • BM-MSCs at 70-80% confluency were either detached with PBS + 0.54 mM Na-EDTA (0.02% Versene) for 20 min at +37°C or were washed once with PBS.
  • the number of detached cells were calculated in Bürker chamber and control (sample Versene 0-) cells were washed four times in cold Ca 2+ -free PBS and frozen as cell pellets at -70°C for mass spectrometric analysis. All centrifugation steps were performed at 300 x g for 5 min.
  • SAT reaction buffer consisting of ⁇ -MEM (Invitrogen), 0.5 % human serum albumin (Albumin SPR, Sanquin), 50 mU rat recombinant ⁇ 2,3-(N)-Sialyltransferase (SAT, Calbiochem) and 1 mg CMP-Neu5Ac (donor) was added to either adherent cells in one ⁇ 10 cm cell culture vessel (sample ⁇ 2,3SAT+Versene) or to detached cells in suspension from one ⁇ 10 cm cell culture vessel transferred to a 24-well cell culture plate (sample Versene+ ⁇ 2,3SAT). The reactions were performed in a humidified 5 % CO2 atmosphere at +37° for 2 hours.
  • the cells in suspension were resuspended every 20 min during the incubation.
  • the enzymatic reactions were stopped by adding excess of cold PBS.
  • the adherent cells were subsequently detached as described previously.
  • Cell viability was determined by Trypan blue exclusion and microscopic analysis in a Bürker chamber for all cells after modifications and detachment.
  • the cells were centrifuged and washing with PBS was repeated additionally 3 times.
  • the cells were finally pelleted by centrifugation and frozen at -70°C for further mass spectrometric N-glycan analysis.
  • the MALDI-TOF mass spectrometric analysis was performed for N-glycosidase F liberated N-glycans essentially as described ( Hemmoranta H. et al., 2007. Exp. Hematol. ).
  • MSC immunophenotype antibodies Plant lectins Glycoform antibodies Positive markers: MAA-FITC Lex GF517 CD90, CD105, CD73 MAA-biotin (2°ab (core 2 O-glycan) sLex HLA-ABC streptavidin FITC) GF526 Negative markers: SNA-FITC (sLex/CSLEX1) GF516, CD14, CD19, CD34, CD45 SNA-biotin (2°ab VPU020 HLA-DR (FITC or PE conjugated) streptavidin FITC) RCA-FITC ECA-biotin (2°ab streptavidin FITC)
  • MAA Maackia amurensis
  • SNA Sambucus nigra
  • RCA Ricinus communis
  • ECA Erythrina cristagalli
  • Viability of BM-MSC after trypsinization in different conditions Number of BM-MSCs in 300 ⁇ l cell suspension. After 2 hours incubation in indicated conditions, the cells still in suspension were collected and their number calculated. The cells adhered to cell culture vessel bottom were not collected. Viability was determined by Trypan blue exclusion. There were less cells left in suspension after 2 h incubation in buffer containing divalent cations ( ⁇ -MEM+0.5%HSA), due to adherence to cell culture vessel if the reaction was not continuously resuspended.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Developmental Biology & Embryology (AREA)
  • Cell Biology (AREA)
  • Hematology (AREA)
  • Rheumatology (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
EP08397517A 2008-07-16 2008-07-16 Cellules modifiées bivalentes Withdrawn EP2166085A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
EP08397517A EP2166085A1 (fr) 2008-07-16 2008-07-16 Cellules modifiées bivalentes
US13/054,541 US9234169B2 (en) 2008-07-16 2009-07-16 Enzymatical modification of cell glycosylation using serum albumin and divalent cations
AU2009272654A AU2009272654B2 (en) 2008-07-16 2009-07-16 Enzymatical modif ication of cell glycosylation using serum albumin and divalent cations
EP09797548A EP2318540A4 (fr) 2008-07-16 2009-07-16 Modification enzymatique de la glycosylation des cellules à l'aide de sérum albumine et de cations divalents
PCT/FI2009/050628 WO2010007214A1 (fr) 2008-07-16 2009-07-16 Modification enzymatique de la glycosylation des cellules à l'aide de sérum albumine et de cations divalents
US14/982,800 US20160215256A1 (en) 2008-07-16 2015-12-29 Enzymatical modification of cell glycosylation using serum albumin and divalent cations

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
EP08397517A EP2166085A1 (fr) 2008-07-16 2008-07-16 Cellules modifiées bivalentes

Publications (1)

Publication Number Publication Date
EP2166085A1 true EP2166085A1 (fr) 2010-03-24

Family

ID=40568564

Family Applications (2)

Application Number Title Priority Date Filing Date
EP08397517A Withdrawn EP2166085A1 (fr) 2008-07-16 2008-07-16 Cellules modifiées bivalentes
EP09797548A Withdrawn EP2318540A4 (fr) 2008-07-16 2009-07-16 Modification enzymatique de la glycosylation des cellules à l'aide de sérum albumine et de cations divalents

Family Applications After (1)

Application Number Title Priority Date Filing Date
EP09797548A Withdrawn EP2318540A4 (fr) 2008-07-16 2009-07-16 Modification enzymatique de la glycosylation des cellules à l'aide de sérum albumine et de cations divalents

Country Status (4)

Country Link
US (2) US9234169B2 (fr)
EP (2) EP2166085A1 (fr)
AU (1) AU2009272654B2 (fr)
WO (1) WO2010007214A1 (fr)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9788539B2 (en) 2011-05-17 2017-10-17 Velico Medical, Inc. Platelet protection solution having beta-galactosidase and sialidase inhibitors
US9609861B2 (en) 2011-05-17 2017-04-04 Velico Medical Inc. Platelet additive solution having a β-galactosidase inhibitor
US20160130324A1 (en) 2014-10-31 2016-05-12 Shire Human Genetic Therapies, Inc. C1 Inhibitor Fusion Proteins and Uses Thereof
MA43270A (fr) 2015-11-19 2021-06-02 Takeda Pharmaceuticals Co Inhibiteur de la c1 estérase humaine recombinante et ses utilisations
JP7195008B2 (ja) 2017-05-26 2022-12-23 ジェノビス エービー グリカン分析のための酵素
US20190060365A1 (en) * 2017-08-25 2019-02-28 Meridigen Biotech Co., Ltd. Pharmaceutical composition for treating chronic obstructive pulmonary disease and method thereof
CN111157736A (zh) * 2018-11-08 2020-05-15 中国科学院大连化学物理研究所 一种基于化学酶促的人血清o糖基化鉴定方法
CN110082415B (zh) * 2019-05-30 2021-01-29 吉林大学 制备基于共轭聚合物纳米粒子的光电化学检测探针及应用

Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002088351A1 (fr) 2001-04-30 2002-11-07 Rbc Biotechnology, Inc. Cellules et organes modifies destines a la xenotransplantation
WO2003105908A2 (fr) 2002-03-15 2003-12-24 Department Of Veterans Affairs, Rehabilitation R & D Service Methodes et compositions faisant appel a des asialodeterminants cellulaires et a des glycoconjugues pour fournir des cellules a des tissus et a des organes
WO2004024761A1 (fr) 2002-09-11 2004-03-25 Fresenius Kabi Deutschland Gmbh Polypeptides-has, notamment, erythropoietine-has ayant subi une acylation
US20040132640A1 (en) 2001-10-10 2004-07-08 Neose Technologies, Inc. Glycopegylation methods and proteins/peptides produced by the methods
WO2004072306A1 (fr) 2003-02-17 2004-08-26 Kiwi Ingenuity Limited Preparation des globules rouges avec un niveau modifie de l'expression de l'antigene du groupe sanguin et leur utilisation dans le controle qualite des reactifs de determination des groupes sanguins
US20050014718A1 (en) 2001-08-20 2005-01-20 Jari Natunen Tumor specific oligosaccharide sequences and use thereof
WO2005014024A2 (fr) 2003-08-08 2005-02-17 Fresenius Kabi Deutschland Gmbh Conjugues de polymere et de proteine lies au moyen d'un groupe de liaison oxime
WO2005014655A2 (fr) 2003-08-08 2005-02-17 Fresenius Kabi Deutschland Gmbh Conjugues d'amidon d'hydroxyalkyle et de proteine
WO2005092391A2 (fr) 2004-03-11 2005-10-06 Fresenius Kabi Deutschland Gmbh Conjugues d'amidon hydroxyalkyle et de proteine, par ligature chimique naturelle
WO2007006864A2 (fr) 2005-07-08 2007-01-18 Suomen Punainen Risti, Veripalvelu Méthode d'évaluation de populations cellulaires
US20070258986A1 (en) 2003-11-19 2007-11-08 Govt of the US as represented by the secretary, Targeted Delivery System for Bioactive Agents
WO2007143204A2 (fr) 2006-06-02 2007-12-13 Sacktein Robert Compositions et procédés pour modifier des glycans de surface cellulaire
WO2008011094A2 (fr) 2006-07-18 2008-01-24 Robert Sackstein Induction par la cytokine de ligands de sélectine sur des cellules
WO2008087260A1 (fr) 2007-01-18 2008-07-24 Suomen Punainen Risti, Veripalvelu Nouveaux agents de liaison cellulaire spécifiques
WO2008087256A1 (fr) 2007-01-18 2008-07-24 Suomen Punainen Risti, Veripalvelu Procédé de modification de cellules

Family Cites Families (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5324663A (en) 1990-02-14 1994-06-28 The Regents Of The University Of Michigan Methods and products for the synthesis of oligosaccharide structures on glycoproteins, glycolipids, or as free molecules, and for the isolation of cloned genetic sequences that determine these structures
WO1991016900A1 (fr) 1990-04-27 1991-11-14 Biogen, Inc. Fucosyle transferases impliquees dans l'expression de molecules d'adhesion
SG49117A1 (en) 1993-03-29 1998-05-18 Kyowa Hakko Kogyo Kk Alfa -1, 3-fucosyltransferase
US20060003924A1 (en) 1994-10-11 2006-01-05 Robert Sackstein Sulfation-independent L-selectin or E-selectin ligand (HCELL) and therapeutics thereof
US5728554A (en) 1995-04-11 1998-03-17 Cytel Corporation Enzymatic synthesis of glycosidic linkages
US5965457A (en) 1995-06-06 1999-10-12 Magnani; John L. Methods of screening for a candidate compound able to bind to CD34+ cells
US5858752A (en) 1995-06-07 1999-01-12 The General Hospital Corporation Fucosyltransferase genes and uses thereof
WO1997032889A1 (fr) 1996-03-08 1997-09-12 The Regents Of The University Of Michigan MURINE α(1,3)FUCOSYLTRANSFERASE Fuc-TVII, ADN LA CODANT, PROCEDE DE PREPARATION, ANTICORPS LA RECONNAISSANT, DOSAGE IMMUNOLOGIQUES PERMETTANT DE LA DETECTER ET PLASMIDES
JP2001507215A (ja) 1997-01-16 2001-06-05 サイテル コーポレイション 組換え糖タンパク質のインビトロでの実用的なシアリル化
WO1998055630A2 (fr) 1997-06-06 1998-12-10 The Governors Of The University Of Alberta α1,3-FUCOSYLTRANSFERASE DE HELICOBACTER PYLORI
US6461835B1 (en) 1998-09-03 2002-10-08 The University Of Oklahoma Fucosyltransferases, polynucleotides encoding fucosyltransferases, and transgenic mammal incorporating same
US6232450B1 (en) 1998-11-12 2001-05-15 The Scripps Research Institute Inhibition of human fucosyltransferases with N-linked Lewis-x and LacNAc analogs
AT408446B (de) 1999-02-18 2001-11-26 Altmann Friedrich Dr Fucosyltransferase-gen
US20050170463A1 (en) * 1999-04-15 2005-08-04 Abraham Bout Recombinant protein production in permanent amniocytic cells that comprise nucleic acid encoding adenovirus E1A and E1B proteins
DE19962204A1 (de) * 1999-12-22 2001-07-05 Cognis Deutschland Gmbh Enzym-katalysierte Modifizierung von Substanzen in biologischen Gemischen
AU2001263149A1 (en) 2000-05-12 2001-11-26 Neose Technologies, Inc. In vitro fucosylation recombinant glycopeptides
FI109811B (fi) 2000-09-26 2002-10-15 Medicel Oy Menetelmä GDP-L-fukoosin valmistamiseksi ja menetelmässä käytettäviä välineitä
US20060210558A1 (en) 2000-10-18 2006-09-21 Robert Sackstein Hematopoietic cell selectin ligand polypeptides and methods of use thereof
DE60140903D1 (de) 2000-10-18 2010-02-04 Brigham & Womens Hospital E-selectin/l-selectin-liganden-polypeptide hämatopoetischer zellen und verfahren zu deren verwendung
CU23007A1 (es) 2001-04-06 2004-12-17 Ct De Inmunologia Molecular Ct De Inmunologia Mole Combinaciones inmunoterapéuticas para el tratamiencombinaciones inmunoterapéuticas para el tratamiento de tumores que sobre-expresan gangliósidos to de tumores que sobre-expresan gangliósidos
FI20011664A (fi) 2001-08-17 2003-02-18 Carbion Oy Syöpäpesifiset oligoskkaridisekvenssit ja niiden käyttö
WO2004017810A2 (fr) 2002-08-20 2004-03-04 Biotie Therapies Corp. Epitopes d'oligosaccharide specifiques de tumeur et leur utilisation
AU2003254139A1 (en) 2002-07-23 2004-02-09 Neose Technologies, Inc. H. Pylori FUCOSYLTRANSFERASES
AU2002950878A0 (en) 2002-08-20 2002-09-12 Proteome Systems Intellectual Property Pty Ltd Method for diagnosing disorders
US7332334B2 (en) 2003-04-18 2008-02-19 Oklahoma Medical Research Foundation Hematopoietic stem cells treated by in vitro fucosylation and methods of use
WO2005017115A2 (fr) 2003-08-11 2005-02-24 Mount Sinai School Of Medicine Of New York University Cellules souches hematopoietiques derivees de sang ombilical
WO2006068720A2 (fr) 2004-11-12 2006-06-29 The Brigham And Women's Hospital, Inc. Polypeptides de ligands de selectine des cellules hematopoietiques et procedes d'utilisation de ces polypeptides
WO2007054620A1 (fr) * 2005-11-08 2007-05-18 Suomen Punainen Risti, Veripalvelu Nouvelles compositions a profils glucidiques tirees de cellules humaines et procedes d'analyse et de modification correspondants
JP4705481B2 (ja) * 2006-01-25 2011-06-22 パナソニック株式会社 窒化物半導体装置

Patent Citations (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002088351A1 (fr) 2001-04-30 2002-11-07 Rbc Biotechnology, Inc. Cellules et organes modifies destines a la xenotransplantation
US20050014718A1 (en) 2001-08-20 2005-01-20 Jari Natunen Tumor specific oligosaccharide sequences and use thereof
US20040132640A1 (en) 2001-10-10 2004-07-08 Neose Technologies, Inc. Glycopegylation methods and proteins/peptides produced by the methods
WO2003105908A2 (fr) 2002-03-15 2003-12-24 Department Of Veterans Affairs, Rehabilitation R & D Service Methodes et compositions faisant appel a des asialodeterminants cellulaires et a des glycoconjugues pour fournir des cellules a des tissus et a des organes
WO2004024761A1 (fr) 2002-09-11 2004-03-25 Fresenius Kabi Deutschland Gmbh Polypeptides-has, notamment, erythropoietine-has ayant subi une acylation
WO2004024776A1 (fr) 2002-09-11 2004-03-25 Fresenius Kabi Deutschland Gmbh Procede de production de derives d'amidon hydroxyalkyle
WO2004072306A1 (fr) 2003-02-17 2004-08-26 Kiwi Ingenuity Limited Preparation des globules rouges avec un niveau modifie de l'expression de l'antigene du groupe sanguin et leur utilisation dans le controle qualite des reactifs de determination des groupes sanguins
WO2005014655A2 (fr) 2003-08-08 2005-02-17 Fresenius Kabi Deutschland Gmbh Conjugues d'amidon d'hydroxyalkyle et de proteine
WO2005014024A2 (fr) 2003-08-08 2005-02-17 Fresenius Kabi Deutschland Gmbh Conjugues de polymere et de proteine lies au moyen d'un groupe de liaison oxime
US20070258986A1 (en) 2003-11-19 2007-11-08 Govt of the US as represented by the secretary, Targeted Delivery System for Bioactive Agents
WO2005092390A2 (fr) 2004-03-11 2005-10-06 Fresenius Kabi Deutschland Gmbh Conjugues d'amidon d'hydroxyalkyle et d'une proteine
WO2005092391A2 (fr) 2004-03-11 2005-10-06 Fresenius Kabi Deutschland Gmbh Conjugues d'amidon hydroxyalkyle et de proteine, par ligature chimique naturelle
WO2007006864A2 (fr) 2005-07-08 2007-01-18 Suomen Punainen Risti, Veripalvelu Méthode d'évaluation de populations cellulaires
WO2007143204A2 (fr) 2006-06-02 2007-12-13 Sacktein Robert Compositions et procédés pour modifier des glycans de surface cellulaire
WO2008011094A2 (fr) 2006-07-18 2008-01-24 Robert Sackstein Induction par la cytokine de ligands de sélectine sur des cellules
WO2008087260A1 (fr) 2007-01-18 2008-07-24 Suomen Punainen Risti, Veripalvelu Nouveaux agents de liaison cellulaire spécifiques
WO2008087256A1 (fr) 2007-01-18 2008-07-24 Suomen Punainen Risti, Veripalvelu Procédé de modification de cellules

Non-Patent Citations (14)

* Cited by examiner, † Cited by third party
Title
BEYER T A ET AL: "ENZYMATIC PROPERTIES OF THE BETA GALACTOSIDE ALPHA-1-2 FUCOSYL TRANSFERASE FROM PORCINE SUBMAXILLARY GLAND", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 255, no. 11, 1980, pages 5373 - 5379, XP002526139, ISSN: 0021-9258 *
EPPLER C M ET AL: "Ganglioside biosynthesis in rat liver characterization of cytidine-5'-monophospho-n-acetylneuraminic acid: Hematoside (Gm3) sialyltransferase", BIOCHIMICA ET BIOPHYSICA ACTA - LIPIDS AND LIPID METABOLISM, ELSEVIER SCIENCE BV. AMSTERDAM, NL, vol. 619, no. 2, 11 August 1980 (1980-08-11), pages 318 - 331, XP023372332, ISSN: 0005-2760, [retrieved on 19800811] *
FREISCHUTZ BETTINA ET AL: "Characterization of sialyltransferase-IV activity and its involvement in the c-Pathway of brain ganglioside metabolism", JOURNAL OF NEUROCHEMISTRY, vol. 64, no. 1, 1995, pages 385 - 393, XP002526141, ISSN: 0022-3042 *
HIDALGO ANDRES ET AL: "Enforced fucosylation of neonatal CD34+ cells generates selectin ligands that enhance the initial interactions with microvessels but not homing to bone marrow", BLOOD, vol. 105, no. 2, 15 January 2005 (2005-01-15), pages 567 - 575, XP002526142, ISSN: 0006-4971 *
KAMINSKA JOANNA ET AL: "Purification and characterization of GDP-L-Fuc: N-acetyl beta-D-glucosaminide alpha1fwdarw6fucosyltransferase from human blood platelets", GLYCOCONJUGATE JOURNAL, vol. 15, no. 8, August 1998 (1998-08-01), pages 783 - 788, XP002526140, ISSN: 0282-0080 *
KANG WJ ET AL., J NUCL MED, vol. 47, pages 1295 - 1301
LAIRSON L L ET AL: "Glycosyltransferases: Structures, functions, and mechanisms", ANNUAL REVIEW OF BIOCHEMISTRY, vol. 77, 14 April 2008 (2008-04-14), pages 521 - 555, XP002526138, ISSN: 0066-4154 *
MIN JJ ET AL., ANN NUCL MED, vol. 20, no. 3, 2006, pages 165 - 70
SACKSTEIN ROBERT ET AL: "Ex vivo glycan engineering of CD44 programs human multipotent mesenchymal stromal cell trafficking to bone", NATURE MEDICINE, vol. 14, no. 2, February 2008 (2008-02-01), pages 181 - 187, XP002526137, ISSN: 1078-8956 *
SACKSTEIN, NATURE MED, 2008
SUJINO ET AL., GLYCOBIOLOGY, vol. 10, no. 3, 2000, pages 313 - 20
TSUKAMOTO H. ET AL., J. BIOL. CHEM, vol. 282, 2007, pages 29794 - 801
XIA ET AL., BLOOD, vol. 104, no. 10, 2004, pages 3091 - 9
XIA LIJUN ET AL: "Surface fucosylation of human cord blood cells augments binding to P-selectin and E-selectin and enhances engraftment in bone marrow", BLOOD, AMERICAN SOCIETY OF HEMATOLOGY, US, vol. 104, no. 10, 15 November 2004 (2004-11-15), pages 3091 - 3096, XP002429129, ISSN: 0006-4971 *

Also Published As

Publication number Publication date
EP2318540A4 (fr) 2011-09-14
US9234169B2 (en) 2016-01-12
EP2318540A1 (fr) 2011-05-11
US20160215256A1 (en) 2016-07-28
AU2009272654B2 (en) 2015-01-15
WO2010007214A1 (fr) 2010-01-21
US20110136203A1 (en) 2011-06-09
AU2009272654A1 (en) 2010-01-21

Similar Documents

Publication Publication Date Title
US20160215256A1 (en) Enzymatical modification of cell glycosylation using serum albumin and divalent cations
US10000734B2 (en) Method for evaluating cell populations
US20100047892A1 (en) Method for modifying cells
AU2008206884B2 (en) Novel methods and reagents directed to production of cells
US20100145032A1 (en) Novel carbohydrate profile compositions from human cells and methods for analysis and modification thereof
AU2008206885B2 (en) Novel carbohydrate from human cells and methods for analysis and modification thereof
JP2010516241A (ja) 新規の特異的細胞結合剤
CA2407707A1 (fr) Modification in vitro de motifs de glycosylation de glycopeptides recombines
US20210123024A1 (en) Compositions and methods for production of exofucosylated cells for clinical applications
Dall'olio et al. α2, 6 sialylation of N‐acetyllactosaminic sequences in human colorectal cancer cell lines. Relationship with non‐adherent growth
JP2010516238A5 (fr)
Zhang et al. Overlapping and unique substrate specificities of ST3GAL1 and 2 during hematopoietic and megakaryocytic differentiation
Takeya et al. Biosynthesis of the blood group P antigen-like GalNAcβ1→ 3Galβ1→ 4GlcNAc/Glc structure: A novel N-acetylgalactosaminyltransferase in human blood plasma
JP2010519922A (ja) ヒト細胞の新規の酸性グリカンマーカ
Class et al. Patent application title: METHOD FOR EVALUATING CELL POPULATIONS
Takeya et al. Biosynthesis of the Blood Group P Antigen-Like 4GlcNAc/Glc Structure: A Novel iV-Acetylgalactosaminyltransferase in Human Blood Plasma1

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MT NL NO PL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL BA MK RS

AKY No designation fees paid
REG Reference to a national code

Ref country code: DE

Ref legal event code: 8566

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20100925